Ethoxyquin mediates lung fibrosis and cellular immunity in BLM-CIA mice by inhibiting HSP90.

BACKGROUND: Patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD) are characterized by severe pulmonary fibrosis and immune dysregulation. Heat shock protein 90 (HSP90) is involved in the progression of pulmonary fibrosis and the immune response. OBJECTIVES: This study aimed to explore whether HSP90 regulates the development of RA-ILD and its underlying mechanism. MATERIAL AND METHODS: In vivo, collagen-induced arthritis (CIA)-mice were treated with bleomycin (BLM) to establish an arthritic mouse model of pulmonary fibrosis. In vitro, human lung fibroblast 1 (HLF1) was exposed to transforming growth factor beta 1 (TGF-ß1) to simulate an RA-ILD model. The RA-ILD models were treated with the HSP90 inhibitor ethoxyquin (EQ) to explore the potential mechanism of HSP90 in RA-ILD. Histopathological analysis was performed, and pulmonary fibrosis was evaluated. The differentiation of M1/M2 macrophages and Th1/Th17/Treg cells was assessed. The role of the TGF-ß/Smad2/3 pathway in EQ-mediated RA-ILD progression was also explored. RESULTS: HSP90α and HSP90ß were upregulated in the RA-ILD models. Ethoxyquin mitigated arthritis in BLM-CIA mice, and reduced the expression of alpha-smooth muscle actin (α-SMA), collagen I (Col-1) and fibronectin (FN), as well as hydroxyproline content, thereby relieving pulmonary fibrosis. In addition, EQ increased M1 macrophages and inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α) levels; conversely, EQ decreased M2 macrophages and vascular endothelial growth factor (VEGF)-A and TGF-ß1 contents. It also decreased Th17 (interleukin (IL)-17) while increasing Th1 (interferon gamma (IFN-γ)) and Treg (Foxp3), and restricted the expression of transforming growth factor beta type receptor I and II (TGF-ßRI and TGF-ßRII) and the phosphorylation of Smad2 and Smad3. CONCLUSIONS: This study revealed that EQ regulated pulmonary fibrosis and cellular immunity by inhibiting HSP90, appearing to act through the TGF-ß/Smad2/3 pathway. These findings suggest that EQ holds potential as a therapeutic agent for treating RA-ILD.

Effects of metformin therapy on HMGB1 levels in rheumatoid arthritis patients.

OBJECTIVE: The traditional treatment of rheumatoid arthritis (RA) has some side effects. We aimed to explore the effect of metformin treatment on the expression of HMGB1, cytokines, T cell subtypes and the clinical outcomes in RA patients. METHODS: The present prospective cohort study recruited 124 RA patients (metformin group) who were treated with metformin and conventional therapy (methotrexate, hydroxychloroquine sulfate and sulfasalazine) and 98 RA patients (conventional therapy group) who were only treated with conventional therapy. All subjects were admitted from December 2018 to December 2021 and continuous medication for 90 days. The serum high mobility group box 1 (HMGB1), tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1ß and C-reactive protein (CRP) levels were measured by enzyme-linked immunosorbent assay (ELISA). Flow cytometric were used to analyze the expression of CD4+ and CD8+. Demographic and clinical statistics including age, body mass index (BMI), sex, course of disease, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), visual analogue score (VAS)and disease activity score (DAS)-28 were collected. RESULTS: The serum levels of HMGB1, CRP, IL-6, CD4+ expression and CD4+/CD8+ ratio were significantly increased in patients with DAS-28 score ≥ 2.6. The serum HMGB1 and cytokines levels of metformin group declined more quickly during the study time. Pearson's analysis supported that a positive correlation existed between the HMGB1 and IL-6, TNF-α, CRP, CD4+, CD4+/CD8+ ratio, and VAS scores. HMGB1 could be a potential diagnostic biomarker for RA patients in active phase. Serum HMGB1 (95% CI 1.133-1.397, P < 0.001) was a factor associated with active RA. CONCLUSION: The serum HMGB1 levels were significantly increased in RA patients in active phase. The serum levels of HMGB1 and inflammatory factors and VAS scores were decreased gradually with metformin treatment. HMGB1 might act as a novel therapeutic target for RA.

MiR-146b-5p targets IFI35 to inhibit inflammatory response and apoptosis via JAK1/STAT1 signalling in lipopolysaccharide-induced glomerular cells.

The dysregulated microRNAs (miRNAs) are implicated in the malignancy of lupus nephritis (LN). This work aims to analyse the effect and mechanism of miR-146b-5p in lipopolysaccharides (LPS)-induced model of LN in vitro. The serum samples of LN patients and normal volunteers were collected. HK-2 cells were challenged via LPS. miR-146b-5p and interferon-induced protein 35 (IFI35) abundances were detected via quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. The inflammatory response was assessed via inflammatory cytokines levels via qRT-PCR and enzyme-linked immunosorbent assay. Cell apoptosis was analysed via flow cytometry and apoptotic protein levels. The protein levels of JAK1/STAT1 signalling were detected via western blot. The relationship of miR-146b-5p and IFI35 was analysed via bioinformatics and dual-luciferase reporter assays. This study revealed that miR-146b-5p level was declined and IFI35 abundance was elevated in serum of LN patients and LPS-challenged HK-2 cells. Functionally, IFI35 overexpression promoted LPS-caused inflammatory response and cell apoptosis, and knockdown of IFI35 caused an opposite trend. Meanwhile, miR-146b-5p targeted IFI35 to suppress inflammatory response and cell inflammatory response and apoptosis via inactivating the JAK1/STAT1 pathway. MiR-146b-5p suppressed inflammatory response and cell apoptosis by IFI35 mediated-JAK1/STAT1 signalling in HK-2 cells, which provided a new mechanism for understanding the pathogenesis of LN.

LncRNA NEAT1 accelerates renal mesangial cell injury via modulating the miR-146b/TRAF6/NF-κB axis in lupus nephritis.

Although growing advances have been made in the regulation of lupus nephritis recently, lupus nephritis is still one of the major causes of death in SLE patients and the pathogenesis remains largely unknown. Therefore, exploring the pathological mechanisms is urgently needed for designing and developing novel therapeutic strategies for lupus nephritis. Human renal mesangial cells (HRMCs) were transfected with sh-NEAT1, miR-146b mimic, pcDNA-NEAT1, miR-146b inhibitor, or sh-TRAF6 to modify their expression. Lipopolysaccharide (LPS) was used to induce inflammatory injury. Cell viability was examined with CCK8. Apoptosis was determined by flow cytometry and Hoechst staining. qRT-PCR and western blot were used to analyze gene expression. The secretion of inflammatory cytokines was examined with ELISA. The bindings of NEAT1 with miR-146b and miR-146b with TRAF6 were tested by dual-luciferase reporter assay. NEAT1 was upregulated in LPS-treated HRMCs. Both the knockdown of NEAT1 and TRAF6 suppressed the LPS-induced inflammatory injury in HRMCs. NEAT1 directly targeted miR-146b to control miR-146b-mediated regulation of TRAF6 expression in HRMCs. NEAT1 promoted the expression of TRAF6 via targeting miR-146b to accelerate the LPS-mediated renal mesangial cell injury in HRMCs. Moreover, TRAF6 activated the NF-κB signaling in HRMCs. NEAT1 accelerated renal mesangial cell injury via directly targeting miR-146b, promoting the expression of TRAF6, and activating the NF-κB signaling in lupus nephritis. Our investigation elucidated novel pathological mechanisms and provided potential therapeutic targets for lupus nephritis.