Rheumatoid arthritis (RA) is a common autoimmune disease worldwide. Neutrophils play critical roles in the onset and development of RA and are the promising target for RA treatment. Tetrandrine is a bis-benzyl isoquinoline alkaloid derived from the traditional Chinese herbal Stephania tetrandra S. Moore. Tetrandrine is effective in alleviating RA by inhibiting macrophage inflammatory response, fibroblast overproliferation, and pannus formation. However, whether tetrandrine regulates the activities of neutrophils in RA is largely unknown. In this study, we adopted adjuvant-induced arthritis (AA) murine model to explore the effect of tetrandrine on RA and neutrophils. Twenty-eight mice were divided into four groups. The control group was injected with PBS in the limbs and treated with PBS by intraperitoneal injection (i.p.) from Day 10 to Day 37. The arthritis murine model was induced by injecting FCA into the ankle joints of hind limbs. The AA group, the AA + TET group, and the AA + DEX group mice were treated with PBS, tetrandrine (6 mg/kg), or dexamethasone (1 mg/kg) i.p. daily, respectively. Arthritic scores were evaluated, and the joint diameter was measured every three days. A cytometric bead assay was performed to measure the concentrations of IFN-γ, TNF-α, and IL-6 in the serum. H&E staining and Safranin O-fast staining were adopted to monitor the tissue changes in the joint. Immunohistochemistry assays were applied to detect the MPO, NE, CitH3, and PAD4 expression levels. To assess the effect of tetrandrine on neutrophil activities in vitro, CCK8 tests were applied to determine cell viability. The qPCR and ELISA were performed to determine IL-1β and IL-6 expression levels. Immunofluorescence assays were performed to measure the formation of NETs. The results indicated that tetrandrine significantly alleviated the symptoms of RA in terms of the ankle diameter (from 4.629 ± 2.729 to 3.957 ± 0.257; ) and ankle score (from 4.000 ± 0.000 to 3.286 ± 0.756; ). Tetrandrine treatment significantly increased the cartilage areas and decreased serum IL-6 significantly (from 5.954 ± 2.127 to 2.882 ± 2.013; ). The immunohistochemistry assays also showed decreased expression levels of NE, MPO, PAD4, and CitH3 induced by tetrandrine in comparison with the AA group ( ). The qPCR assays and ELISAs showed that tetrandrine had an anti-inflammatory effect in vitro by significantly inhibiting IL-6 ( ). The immunofluorescence assays showed that NET formation induced by PMA could be reduced by tetrandrine ( ). In conclusion, tetrandrine has good efficacy in treating RA by regulating neutrophil-involved inflammation and NET formation.

The most characteristic feature of membranous nephropathy (MN) is the presence of subepithelial electron dense deposits and the consequential thickening of the glomerular basement membrane. There have been great advances in the understanding of the destiny of immune complexes in MN by the benefit of experimental models represented by Heymann nephritis. Subepithelial immune complexes are formed in situ by autoantibodies targeting native autoantigens or exogenous planted antigens such as the phospholipase A2 receptor (PLA2R) and cationic BSA respectively. The nascent immune complexes would not be pathogenic until they develop into immune deposits. Podocytes are the major source of autoantigens in idiopathic membranous nephropathy. They also participate in the modulation and removal of the immune complexes to a large extent. The balance between deposition and clearance is regulated by a wide range of factors such as the composition and physicochemical properties of the immune complexes and the complement system. Complement components such as C3 and C1q have been reported to be precipitated with the deposits whereas a complement regulatory protein CR1 expressed by podocytes is involved in the phagocytosis of immune complexes by podocytes. Podocytes regulate the dynamic change of immune complexes which is disturbed in membranous nephropathy. To elucidate the precise fate of the immune complexes is essential for developing more rational and novel therapies for membranous nephropathy.

Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is known as a rare disease characterized by inflammatory lesions on bones and skin. Polymorphism of clinical manifestation and lack of molecular biomarkers have both limited its diagnosis. Our study performed RNA sequencing (RNA-seq) and integrative bioinformatics analysis of long noncoding RNA (lncRNA)-messenger RNA (mRNA) profile in patients with SAPHO syndrome and healthy controls. A total of 4,419 differentially expressed (DE) mRNAs and 2,713 lncRNAs were identified (p < 0.05, fold change > 2) and a coexpression network was constructed to further investigate their regulatory interactions. The DE lncRNAs were predicted to interact with mRNAs in both cis and trans manners. Functional prediction found that the lncRNA-targeted genes may function in SAPHO syndrome by participating in biological process such as adipocytokine signaling pathway, ErbB signaling pathway, FoxO signaling pathway, as well as production and function of miRNAs. The expression levels of three pairs of coexpressed lncRNA-mRNAs were validated by qRT-PCR, and their relative expression levels were consistent with the RNA-seq data. The deregulated RNAs GAS7 and lnc-CLLU1.1-1:2 may serve as potential diagnostic biomarkers, and the combined receiver operating characteristic (ROC) curve of the two showed more reliable diagnostic ability with an AUC value of 0.871 in distinguishing SAPHO patients from healthy controls. In conclusion, this study provides a first insight into long noncoding RNA transcriptome profile changes associated with SAPHO syndrome and inspiration for further investigation on clinical biomarkers and molecular regulators of this inadequately understood clinical entity.

Objective: Si Miao San is effective in ameliorating rheumatoid arthritis (RA) both clinically and experimentally. NETs play a fundamental role in the onset and progression of RA. The goal of this study was to explore the therapeutic effects of Si Miao San (SMS) on adjuvant-induced arthritis in mice and the regulatory mechanisms of NETs both in vivo and in vitro. Methods: SMS decoctions were identified using LC-MS/MS analysis and TCMSP. In the adjuvant induced RA murine model, SMS decoction and methotrexate were administered orally. Disease progression was analysed by assessing arthritic scores and joint diameter, H&E staining, safranin O-fast green staining, toluidine blue staining and micro-CT analysis. The expression of NE, MPO, PAD4, LC3B, CitH3, p-AKT and p-PI3K and the production of ROS were detected using IHC, WB and IF analyses. Cytokines in the sera of the mice were detected using cytometric bead arrays. After the in vitro culture of neutrophils, NE, MPO, PAD4, LC3B, CitH3, ROS, p-PI3K and p-AKT were measured using IF and WB analyses. Autophagy was further observed with TEM. Results: SMS decoction compounds were first identified. Compared with the model group, SMS significantly inhibited joint swelling, inflammation progression and bone destruction. The levels of NE, MPO, PAD4, CitH3, LC3B, ROS production and relative expression of p-AKT and p-PI3K in joint tissues were significantly reduced in the SMS group compared to the model group (P < 0.05). In vitro culture, SMS-containing serum significantly reduced the LC3B-II/LC3B-I ratio and the relative expression levels of p-AKT and p-PI3K, as well as the levels of ROS, NE, MPO, PAD4, and CitH3 compared with those in the PMA-treated group (P < 0.05), which was abolished by the treatment with the AKT activator SC79. Conclusion: The SMS-induced suppression of inflammation in experimental RA occurred through the modulation of the AKT/ROS/autophagy axis.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease that mainly affects the joints. Ya Han Jie (YHJ) is a Dai medicine that has significant therapeutic effects on RA. However, the bioactive compounds and potential mechanisms of YHJ in treating RA still need to be elucidated. Purpose: The aim of this study was to investigate the effects of YHJ on RA and to illuminate its underlying therapeutic mechanisms. Methods/study design: We injected Freund's complete adjuvant (FCA) into the joint cavity of mice to establish an adjuvant-induced arthritis (AA) mouse model and evaluate the therapeutic effect and safety of YHJ on RA. First, we measured the joint diameters of the mice and assessed the effect of YHJ on the degree of joint swelling. Second, we assessed the effects of YHJ on pathological changes in the joints of AA mice by performing Hematoxylin-eosin staining and Safranin O-fast green staining. Afterward, we used network pharmacology, molecular docking, and molecular dynamics simulations to identify the active components in YHJ that act on RA and explored the therapeutic mechanism of YHJ in treating RA with RNA sequencing. Moreover, we evaluated the effect of YHJ on neutrophil extracellular traps (NETs) using ankle joint tissue sections and purified neutrophils. Finally, we further evaluated the effects of YHJ on the gut microbiota of AA mice by 16S rDNA sequencing. Results: YHJ significantly reduced joint swelling, arthritis scores, and synovitis-related pathological changes in AA mice. YHJ also inhibited the expression of TNF-α, IL-6, IL-17A, and IFN-γ. UPLC-Q-TOF MS/MS was used to characterize the chemical composition of YHJ, identifying 104 compounds mainly classified as amino sugars, glycosides, alkaloids, and terpenoids. Network pharmacology was employed to identify potential targets and signaling pathways of YHJ in the treatment of RA, and transcriptomic analysis revealed that YHJ downregulated genes associated with NET formation. In vivo and in vitro experiments further confirmed that YHJ significantly inhibited NET formation. Conclusion: YHJ has a significant therapeutic effect on AA mice by reducing the phosphorylation and activation of NF-κB, decreasing the expression of proteins related to NET formation, and inhibiting NET formation and the release of inflammatory factors in vitro and in vivo.