LncRNA XIST/miR-381-3P/STAT1 axis as a potential biomarker for lupus nephritis.

OBJECTIVE: We aim to investigate the potential roles of key genes in the development of lupus nephritis (LN), screen key biomarkers, and construct the lncRNA XIST/miR-381-3P/STAT1 axis by using bioinformatic prediction combined with clinical validation, thereby providing new targets and insights for clinical research. METHODS: Gene expression microarrays GSE157293 and GSE112943 were downloaded from the GEO database to obtain differentially expressed genes (DEGs), followed by enrichment analyses on these DEGs, which were enriched and analyzed to construct a protein-protein interaction (PPI) network to screen core genes. The lncRNA-miRNA-mRNA regulatory network was predicted and constructed based on the miRNA database. 37 female patients with systemic lupus erythematosus (SLE) were recruited to validate the bioinformatics results by exploring the diagnostic value of the target ceRNA axis in LN by dual luciferase and real-time fluorescence quantitative PCR (RT-qPCR) and receiver operating characteristic (ROC). RESULTS: The data represented that a total of 133 differential genes were screened in the GSE157293 dataset and 2869 differential genes in the GSE112943 dataset, yielding a total of 26 differentially co-expressed genes. Six core genes (STAT1, OAS2, OAS3, IFI44, DDX60, and IFI44L) were screened. Biological functional analysis identified key relevant pathways in LN. ROC curve analysis suggested that lncRNA XIST, miR-381-3P, and STAT1 could be used as potential molecular markers to assist in the diagnosis of LN. CONCLUSION: STAT1 is a key gene in the development of LN. In conclusion, lncRNA XIST, miR-381-3P, and STAT1 can be used as new molecular markers to assist in the diagnosis of LN, and the lncRNA XIST/miR-381-3P/STAT1 axis may be a potential therapeutic target for LN.

[High expression of anti-ß2 glycoprotein I (ß2GPI) autoantibody exacerbates inflammation and immune dysfunction in connective tissue disease].

Objective To observe the expression of anti-ß2 glycoprotein I (ß2GPI) autoantibody in connective tissue diseases and its relationship with the degree of inflammation and immune function. Methods Patients with broad connective tissue diseases including connective tissue disease (CTD), rheumatoid arthritis (RA), Sjogren's syndrome (SS), and systemic lupus erythematosus (SLE) were observed. ß2GPI was quantified by chemiluminescence, ESR was measured by Weil's method, and C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated polypeptide (CCP) antibody were measured by automatic biochemical analyzer. Results ß2GPI and their subtypes were significantly higher in RA patients compared with CTD, SS, and SLE patients. CRP was positively associated with anti-ß2GPI antibody and anti-ß2GPI antibody IgM in patients with connective tissue disease. ESR was positively associated with anti-ß2GPI antibody. Anti-ß2GPI antibody and anti-ß2GPI antibody IgM were elevated in the abnormal CRP group compared with the normal CRP group. Compared with the ESR normal group, anti-ß2GPI antibody and anti-ß2GPI antibody IgG were elevated in the ESR abnormal group. Anti-ß2GPI antibody was positively correlated with ESR and anti-CCP antibody in RA patients. Anti-ß2GPI antibody IgG was positively correlated with RF. Conclusion ß2GPI can be used as a predictor of the degree of inflammation and assessment of immune disorders in CTD.

Xinfeng Capsule Inhibits Pyroptosis and Ameliorates Myocardial Injury in Rats with Adjuvant Arthritis via the GAS5/miR-21/TLR4 Axis.

Purpose: This study probed the mechanism of action of Xinfeng Capsule (XFC) in myocardial injury in rats with adjuvant arthritis (AA) via the growth arrest-specific transcript 5 (GAS5)/microRNA-21 (miR-21)/Toll-like receptor 4 (TLR4) axis. Methods: Rats were injected with Freund's complete adjuvant to establish a rat model of AA. Then, some modeled rats were given normal saline or drugs only, and some modeled rats were injected with adeno-associated viruses or necrosulfonamide (NSA; a pyroptosis inhibitor) before drug administration. Toe swelling and arthritis index (AI) were calculated. Pathological and morphological changes in synovial and myocardial tissues were analyzed with hematoxylin-eosin staining, and pyroptotic vesicles and the ultrastructural changes of myocardial tissues were observed with transmission electron microscopy. The serum levels of interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor (TNF)-α were detected, and lactate dehydrogenase (LDH) release was measured in myocardial tissues, accompanied by the examination of GAS5, miR-21, TLR4, nuclear factor-kB (NF-κB) p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), Caspase-1, and Gasdermin D (GSDMD) expression in myocardial tissues. Results: After AA modeling, rats presented with significantly increased toe swelling and AI scores, synovial and myocardial tissue damage, elevated pyroptotic vesicles, and markedly enhanced serum levels of IL-1ß, IL-18, IL-6, and TNF-α, accompanied by significantly diminished GAS5 expression, substantially augmented miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression, greatly increased LDH release in myocardial tissues. XFC treatment significantly declined toe swelling, AI scores, synovial and myocardial tissue damage, and the serum levels of IL-1ß, IL-18, IL-6, and TNF-α in AA rats. Additionally, XFC treatment markedly elevated GAS5 expression and substantially lowered LDH release and miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression in myocardial tissues of AA rats. Moreover, the above effects of XFC in AA rats were further promoted by GAS5 overexpression or NSA treatment. Conclusion: XFC alleviated myocardial injury in AA rats by regulating the GAS5/miR-21/TLR4 axis and inhibiting pyroptosis and pro-inflammatory cytokine secretion.

[Circular RNA Cbl proto-oncogene B (circCBLB) inhibits proliferation, promotes apoptosis, and increases anti-inflammatory cytokine levels of fibroblast-like synoviocytes in rheumatoid arthritis].

Objective To explore the regulatory axis of circular RNA Cbl proto-oncogene B (circCBLB)/miR-486-5p on the proliferation, apoptosis, and inflammatory cytokines of fibroblast-like synoviocytes in rheumatoid arthritis (RA-FLS). Methods Human RA-FLS were stimulated with 100 µL of 10 ng/mL of tumor necrosis factor-alpha (TNF-α) to establish the model. The binding relationship of circCBLB/miR-486-5p was validated by a dual-luciferase reporter gene assay. pcDNA3.1/siRNA-circCBLB, negative control (pcDNA3.1-NC/si-NC), and miR-486-5p-mimics were created and transfected into RA-FLS, respectively. The experiment was divided into seven groups: control, TNF-α-treated RA-FLS, pcDNA3.1-circCBLB, pcDNA3.1-NC, si-circCBLB, si-NC, and pcDNA3.1-circCBLB combined with miR-486-5p-mimics. Cell viability was assessed by a CCK-8 assay; cell cycle and apoptosis by flow cytometry; colony formation ability by a colony formation assay; and the expression levels of circCBLB and miR-486-5p by real-time quantitative PCR. The levels of interleukin 4 (IL-4), IL-10, IL-6 and TNF-α were measured by ELISA. Results The dual-luciferase reporter gene assay showed that circCBLB bound to the 3' untranslated region (3'UTR) of miR-486-5p. Compared with the model group at the same time point, the cell viability of the overexpression group was lower, while that of the interference group was higher. Compared with the model group, the overexpression group had a higher apoptosis rate, a higher proportion in S and G2 phases, a lower colony formation rate, a lower miR-486-5p expression level, higher IL-4 and IL-10 levels, and lower IL-6 and TNF-α levels. The interference group had a lower apoptosis rate, a lower proportion in S and G2 phases, a higher colony formation rate, a higher miR-486-5p expression level, and a higher TNF-α level. The pcDNA3.1-circCBLB combined with miR-486-5p-mimics group reversed the effects of circCBLB on cell viability, apoptosis rate, cell cycle, colony formation ability, antiinflammatory cytokines, and proinflammatory cytokines. Conclusion circCBLB inhibits the viability of RA-FLS, increases apoptosis rate, prolongs the cell cycle, reduces colony formation ability, increases antiinflammatory cytokines, and decreases proinflammatory cytokines. In contrast, miR-486-5p has opposite regulatory effects on circCBLB and can partially reverse and offset the effects of circCBLB.

Chonggu Granules Improve Cartilage Matrix Metabolism in Knee Osteoarthritis via the miR-148a-3p/Wnt/ß-Catenin Pathway.

Purpose: This study aims to explore the effect and underlying mechanism of Chonggu Granules (CGG) in knee osteoarthritis (KOA) in rats. Methods: A papain-induced KOA model was established in rats. The pathological alterations of extracellular matrix in rat cartilage tissues were observed through hematoxylin and eosin (H&E) staining, followed by Mankin score for quantitative scoring. The ultrastructure of cartilage extracellular matrix was examined under a transmission electron microscopy (TEM). ELISA was used to measure the levels of IL-6, TNF-α, and IL-1ß in rat serum. Immunofluorescence was performed for assessing the levels of MMP-3, MMP-13, and Col2al in rat cartilage. Western blot was used to identify the protein expressions of wnt1, GSK-3ß, ß-catenin, and Aggrecan in rat cartilage. The mRNA relative expressions of miR-148a-3p, wnt1, ß-catenin, and GSK-3ß in rat cartilage were detected by RT-PCR. Luciferase reporter gene was used to detect the target genes of miR-148a-3p. Results: CGG significantly improved articular cartilage tissue and extracellular matrix metabolism compared to the model group as indicated by H&E, Mankin score, and TEM data. Moreover, low, medium, and high doses of CGG reduced the levels of IL-6, TNF-α, IL-1ß, MMP-3, and MMP-13 in serum to varying degrees but increased the levels of Col2al and Aggrecan. Mechanistically, CGG targeted wnt1 by increasing the expression of miR-148a-3p in a dose-dependent manner, thereby downregulating the mRNA and protein expressions of ß-catenin in cartilage tissue and upregulating the mRNA and protein expressions of GSK-3ß. Conclusion: CGG may control the miR-148a-3p/wnt/ß-catenin signaling pathway to decrease the levels of its downstream target genes MMP-13 and MMP-3, increase the expressions of Col2al and Aggrecan, and downregulate the contents of inflammatory cytokines IL-6, TNF-α, and IL-1ß, thereby improving the metabolism of cartilage extracellular matrix and alleviating the degeneration of articular cartilage in KOA.

A novel pharmaceutical preparation of Tripterygium wilfordii Hook. f. regulates macrophage polarization to alleviate inflammation in rheumatoid arthritis.

OBJECTIVES: To validate the enhanced therapeutic effect of Tripterygium wilfordii Hook. f. (TWHF) in the treatment of rheumatoid arthritis (RA) by restoring homeostasis of M1/M2 macrophages. METHODS: This study, using random walk models and network pharmacology, examined the molecular targets and mechanism of TWHF in RA. Based on clinical observations and experiments in arthritis animal models, the effects of TWHF on macrophage polarization, related signal pathways, and targets were examined. Triptolide, a component of TWHF, was used to intervene arthritis rats. KEY FINDINGS: Network pharmacological analysis revealed the key RA target genes related to TWHF. TWHF showed a strong correlation with the improvement of inflammatory indicators. TWHF inhibited the factors secreted by M1 macrophages such as IL-1ß, IL-6, CXCL8, TNF-α, and VEGF-A, but promoted IL-10 from M2 macrophages. Quantitative liquid-phase chip assay showed that triptolide reduced the levels of TNF-α, CXCL2, and VEGF, while IL-4 and IL-10 were increased in arthritis model. Meanwhile, triptolide inhibited the NF-κB, PI3K/AKT, and p38 MAPK signaling pathways, which in turn improved the RA joint inflammation and fixed immune imbalance. CONCLUSIONS: Triptolide downregulate the expression of M1 macrophage-secreted factors that inhibit the overactivation of inflammatory signaling pathways.

Microstructure and High-Temperature Ablation Behaviour of Hafnium-Doped Tungsten-Yttrium Alloys.

W is a widely used refractory metal with ultra-high melting point up to 3410 °C. However, its applications are limited by poor ablation resistance under high-temperature flame and air flow, which is crucial for aerospace vehicles. To improve the ablation resistance of W under extreme conditions, W-Y alloys doped with different Hf mass fractions (0, 10, 20, and 30) were prepared using the fast hot pressing sintering method. Microstructure and ablation behaviours at 2000 °C were investigated. Results showed that adding an appropriate amount of Hf improved the properties of the W-Y alloy evidently. In particular, the hardness of the alloy increased with the increased content of Hf. The formation of the HfO2 layer on the surface during ablation decreased the mass and linear ablation rates, indicating enhanced ablation resistance. However, excessive Hf addition will result in crack behaviour during ablation. With a Hf content of 20 wt.%, the alloy exhibited high stability and an excellent ablation resistance.

Role of m6A modification and novel circ_0066715/ miR-486-5p/ ETS1 axis in rheumatoid arthritis macrophage polarization progression.

Rheumatoid arthritis (RA) is a systemic disease dominated by inflammatory synovitis. RA synovial macrophages tend undergo M1-type macrophage polarization. Then, polarized M1-type macrophages secrete abundant pro-inflammatory cytokines, causing joint and cartilage destruction. N6-methyladenosine (m6A) methylation modification, circular RNA (circRNA), microRNA (miRNA), messenger RNA (mRNA), etc. are involved in the inflammatory response of RA. We found that there is an imbalance of inflammatory polarization in RA, which is manifested by a sharp increase in inflammatory markers and a high inflammatory response. Here, we show that RA was closely associated with low expression of circ_0066715. The overexpression of circ_0066715 significantly increased the ETS1 levels in RA-FLS cells, decreased cytokine secretion by M1-type macrophages, elevated M2-type cytokines, and inhibited FLS proliferation. Interestingly, the overexpression of miR-486-5p significantly suppressed the attenuation of the cell function and the effect on M1 macrophage polarization caused by circ_0066715 positive expression. WTAP may be involved in the methylation process of ETS1 in RA. ETS1 m6A methylation levels were altered upon WTAP intervention. The overexpression or interference of circ_0066715 decreased or increased WTAP expression. Our findings provide a novel circRNA/miRNA/mRNA regulatory axis and m6A regulatory mechanism involved in the process of RA macrophage polarization, thereby providing a powerful diagnostic and therapeutic strategy for RA treatment.

Comprehensive Analysis and Functional Characteristics of Differential Expression of N6-Methyladenosine Methylation Modification in the Whole Transcriptome of Rheumatoid Arthritis.

N6-methyladenosine (m6A) modification is the most prevalent chemical modification in eukaryotic mRNA and is associated with the development of various immune diseases. However, the role of m6A methylation in rheumatoid arthritis (RA) development is unclear. We preliminarily explored the role of m6A methylation-related mRNAs in RA for its clinical application. The discovery of m6A methylation-modifying genes in this study may provide a fresh perspective on the development of drugs for RA treatment. High-throughput sequencing combined with methylated RNA immunoprecipitation (MeRIP-seq) and RNA sequencing were used to assess whole-transcriptome m6A modifications in the synovium of patients with RA. The relationship between m6A-modified target genes and RA inflammation and macrophages was determined. The expression of the m6A-modified significant transcript-enriched inflammatory signaling pathway was assessed through animal experiments. Differentially expressed m6A genes were correlated with macrophage activation involved in immune response, vascular endothelium, MAPK signaling pathway, PI3K - Akt signaling pathway, and other inflammatory processes. Furthermore, combined analysis with m6A-seq and RNA-seq revealed 120 genes with significant changes in both m6A modification and mRNA expression. We selected the top 3 candidate mRNAs that were upregulated and downregulated simultaneously. The expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) mRNA and protein in RA patients was lower than that in healthy control (HC). SHC-binding protein 1 (SHCBP1) and neurexophilin-3 (NXPH3) mRNA expressions were increased in RA patients. The expression of M1 macrophages was increased in RA patients. RA markers are such as rheumatoid factor (RF) and peptide containing citrulline (CCP). Further animal experiments showed that the expression of synovial MAPK, PI3K, and Akt1 proteins in the RA model was increased, and the PTEN, p-PTEN protein expression was decreased. PI3K, Akt1, PTEN, and p-PTEN were correlated to RA joint inflammation. This study revealed a unique pattern of differential m6A methylation modifications in RA and concluded that m6A modification is related to the occurrence of RA synovial inflammation.

Role of an Ice Surface in the Photoreaction of Coumarins.

Ice affects many chemical reactions in nature, which greatly influences the atmosphere, climate, and life. However, the exact mechanism of ice in these chemical reactions remains elusive. For example, it is still an open question as to whether ice can act as a catalyst to greatly enhance the reactivity and selectivity, which is essential for the production of some natural compounds in our planet. Here, we discover that ice can lead to high efficiency and stereoselectivity of the [2 + 2] photodimerization of coumarin and its derivatives. The conversion of the [2 + 2] photodimerization of coumarins enhanced by ice is dozens of times higher than that in the unfrozen saturated solution, and the reaction displays a high syn-head-head stereoselectivity (>95%) in comparison with those in the absence of the ice. Note that almost no reaction occurs in the crystal powder and melt of the coumarins, indicating that the role of ice in the photodimerization reaction is not simply due to the usual mechanisms found in the freezing concentration. We further reveal that the reaction rate is found to be proportional to the total area of the ice surface and follows Michaelis-Menten-like kinetics, indicating that the ice surface catalyzes the reaction. Molecular dynamics simulations demonstrate that ice surfaces can induce reactants to form a two-dimensional liquid-crystal-ordered layer with a suitable intermolecular distance and unique side-by-side packing, facilitating stereoselective photodimerization for syn-head-head dimers. These findings give evidence that ice-surface-induced molecular assembly may play an important role in atmospheric heterogeneous photoreaction processes.

Yunpi Qufeng Chushi Formula for Pre-Rheumatoid Arthritis: Study Protocol for a Multiple-Center, Double-Blind, Placebo-Controlled Randomized Controlled Trial.

Introduction: Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive bone erosion on diarthrodial joints. RA patients usually experienced three stages before final diagnosis: the health period, the pre-clinical period (immune response exists without clinical symptoms), and the pre-RA period (immune response exists with mild inflammatory manifestation). Presently, there is seldom guidance referring to early intervention which is a benefit for stable disease conditions and low morbidity. Prophylactic treatment is a major feature of traditional Chinese medicine (TCM). In this present study, a multi-center, double-blind, placebo-controlled clinical trial is carried out to evaluate both efficacy and safety in preventing RA progression on Yunpi Qufeng Chushi formula (YQCF). Method: The multi-center, double-blind, placebo-controlled clinical trial is conducted in 13 hospitals nationwide. A total of 390 patients ages between 18 and 70 will be recruited in the trial. They will be randomly assigned to the intervention group (YQCF) and placebo group. The follow-up visit will be taken every 3 months from baseline to 1 year. Diagnosis, disease activity scores, clinical disease activity index (CDAI), simplified disease activity index (SDAI), TCM syndrome scores, and safety assessments will be recorded at every visit. Joint color doppler ultrasound, health assessment questionnaire-disability index (HAQ-DI), and functional assessment of chronic illness therapy-fatigue (FACIT-F) will be recorded at baseline and the last visit. Discussion: This work will provide evidence of YQCF in preventing RA progression. However, whether early intervention would benefit the controlling RA disease still needs a long-term follow-up. Ethics and dissemination: Protocol version 2 (201910-1). This research was approved by the medical ethics committee of Zhejiang Chinese Medical University (2019-045). Results will be published in a peer-reviewed academic journal. Trial registration numbers: http://www.chictr.org.cn/index.aspx, ChiCTR1900024166.

Wilforlide A ameliorates the progression of rheumatoid arthritis by inhibiting M1 macrophage polarization.

Rheumatoid arthritis (RA) is an autoimmune disease with increased M1 macrophages. The classical activated M1 macrophages produce various cytokines to control inflammation. Wilforlide A is a natural product that displays anti-inflammatory activities. However, the effect of Wilforlide A on RA progression and the potential mechanisms are unclear. Herein, the collagen-induced arthritis (CIA) mouse was used as an experimental model of RA. The administration of Wilforlide A reduced clinical scores, joint swelling and histological damage in ankle joints of RA mice. The secreted pro-inflammatory factors (MCP1, GM-CSF and M-CSF) and M1 biomarker iNOS in synovium were inhibited by Wilforlide A. In vitro, macrophages deriving from THP-1 cells were stimulated with LPS/IFN-γ to mimic M1 polarization. Similarly, Wilforlide A blocked macrophages polarizing towards M1 subsets. The in vitro results demonstrated that Wilforlide A suppressed LPS/IFN-γ-induced TLR4 upregulation, IκBα degradation and NF-κB p65 activation. In addition, TAK242 (a TLR4 inhibitor) treatment caused a similar inhibitory effect on M1 polarization with Wilforlide A, whereas it was less than the combination of TAK242 and Wilforlide A. Therefore, this work supports that Wilforlide A ameliorates M1 macrophage polarization in RA, which is partially mediated by TLR4/NF-κB signaling pathway inactivation.

The protective effects of qihuang jianpi zishen decoction on mrl/lpr mice and its mechanism.

Systemic lupus erythematosus (SLE) is a chronic disease of the autoimmune system with multiple damages, most commonly renal damage. The aim of the present study is to examine the therapeutic ability of Qihuang Jianpi Zishen decoction (QJZ) on MRL/lpr mice and uncover its mechanism preliminarily. Twenty-four female MRL/lpr mice were assigned into the model, prednisolone, mycophenolate mofetil and QJZ groups randomly. Six C57BL/6 mice were considered as controls. Each group was treated with corresponding drugs for 4 weeks, anti-dsDNA autoantibodies, C3 and C4, renal function and renal histopathological changes were observed. The expression of GAS5/miR-21/sprouty1 axis and ERK/CREB pathway in kidney was identified by western blotting and qRT-PCR. Compared with MRL/lpr mice, anti-dsDNA autoantibodies of mice treated with QJZ were significantly down-regulated, C3 and C4 were significantly up-regulated. QJZ also alleviated proteinuria, decreased SCr and BUN levels and minimized renal histopathological changes. In addition, QJZ affected the expression of GAS5/miR-21/sprouty1 axis and the phosphorylation of ERK/CREB pathway in renal tissues. QJZ bears therapeutic ability on healing renal injury in MRL/lpr mice. These effects may be achieved by regulating the GAS5/miR-21/sprouty1 axis and inhibiting the ERK/CREB pathway, thus improving the excessive proliferation of glomerular mesangial cells.

Characterization and Microstructural Evolution of Continuous BN Ceramic Fibers Containing Amorphous Silicon Nitride.

Boron nitride (BN) ceramic fibers containing amounts of silicon nitride (Si3N4) were prepared using hybrid precursors of poly(tri(methylamino)borazine) (PBN) and polycarbosilane (PCS) via melt-spinning, curing, decarburization under NH3 to 1000 °C and pyrolysis up to 1600 °C under N2. The effect of Si3N4 contents on the microstructure of the BN/Si3N4 composite ceramics was investigated. Series of the BN/Si3N4 composite fibers containing various amounts of Si3N4 from 5 wt% to 25 wt% were fabricated. It was found that the crystallization of Si3N4 could be totally restrained when its content was below 25 wt% in the BN/Si3N4 composite ceramics at 1600 °C, and the amorphous BN/Si3N4 composite ceramic could be obtained with a certain ratio. The mean tensile strength and Young's modulus of the composite fibers correlated positively with the Si3N4 mass content, while an obvious BN (shell)/Si3N4 (core) was formed only when the Si3N4 content reached 25 wt%.

[Mechanism of Xinfeng Capsules improving rheumatoid arthritis based on CD19~+B cells regulating FAK/CAPN/PI3K pathway].

To observe the effect of Xinfeng Capsules on rheumatoid arthritis (RA) B lymphocytes,inflammatory mediators,FAK/CAPN/PI3K pathway,in order to explore the mechanism of Xinfeng Capsules in improving clinical symptoms of RA.Joint and systemic symptoms of RA patients were observed,and laboratory indicators[hemoglobin (HGB),platelet count (PLT),erythrocyte sedimentation (ESR),immunoglobulin (Ig) G,Ig A,Ig M,rheumatoid factor (RF),anti-cyclic citrulline antibody (CCP-AB),C-reactive protein (CRP)]were detected.ELISA was used to detect serum interleukin (IL)-1ß，IL-10,IL-33,chemokine 5 (CCL5),and vascular endothelial growth factor (VEGF).CD3~-CD19~+B cells were measured by flow cytometry.Western blot was used to detect FAK,p-FAK,CAPN,PI3K protein.The results showed that Xinfeng Capsules could significantly alleviate RA joint and systemic symptoms and improve clinical efficacy.And Xinfeng Capsules could increase HGB,decrease PLT,CCP-AB,CRP,ESR index,upregulate IL-10 expression,and down-regulate IL-1ß，IL-33,CCL5,VEGF,CD3~-CD19~+B cells,FAK,p-FAK,CAPN,PI3K expressions (P<0.01).Based on the above results,Xinfeng Capsules may reduce the expression of CD3~-CD19~+,regulate the balance of inflammatory cytokines and chemokines,inhibit abnormal activation of FAK/CAPN/PI3K pathway,and improve clinical symptoms of RA.

[Inflammation caused by different immune cell subsets is involved in bone destruction of rheumatoid arthritis].

Objective To observe the relationship of rheumatoid arthritis (RA) bone destruction with T cells, regulatory T cells (Tregs), CD19+ B cells and immune inflammation. Methods The study enrolled randomly 20 RA patients (RA group) and 20 normal controls (NC group). Bone mineral density was measured by DXEA dual energy X-ray bone densitometer. The serum levels of γ-interferon (IFN-γ) of Th1 cells, interleukin-4 (IL-4) of Th2 cells, IL-17, type I procollagen amino terminal propeptide (PINP), type I collagen carboxy terminal peptide (CTx), receptor activator of NF-κB ligand (RANKL) and osteoprotectin (OPG) were detected by ELISA. T-cell subsets, Tregs, CD19+ B cells in peripheral blood were measured by flow cytometry. Spearman method was used to analyze the correlations of T cells, Treg and CD19+ B cells with bone density and Th1 and Th2 cytokines. Results The bone mineral density T value of the RA group was lower than that of the NC group. The bone mineral density T value of the double forearm was lower than that of the lumbar spine in the RA group. Compared with the NC group, the expression of IFN-γ, IL-17, Th1/Th2 cells, CTx, RANKL increased, and IL-4, PINP decreased in the RA group. Compared with the NC group, the expression of CD4+/CD8+ T cells, CD19+ B cells increased, and CD8+ T cells, CD4+CD25+ Tregs decreased in the RA group. Correlation analysis showed that CD8+ T cells were positively correlated with Th1/Th2 cells and IL-17, while CD4+ CD25+ Tregs were negatively correlated with CTx, and CD19+ B cells were negatively correlated with OPG in RA. Conclusion T cells and CD19+ B cells may participate in the process of bone destruction in RA by mediating inflammatory immunity.

[Decreased long-chain non-coding RNA MALAT1 expression and increased hsa-miR155-3p expression involved in Notch signaling pathway regulation in rheumatoid arthritis patients].

Objective To observe the expression of long-chain non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and hsa-miR155-3p in patients with rheumatoid arthritis (RA) and their relationship with Notch signaling pathway, and to explore the possible pathogenesis of RA. Methods Peripheral blood of RA patients (RA group) and healthy controls (NC group) were used to screen differentially expressed lncRNA and mRNA by high-throughput gene sequencing. Reverse-transcription PCR was used to detect the expression of lncRNA MALAT1, hsa-miR155-3p and Notch signaling pathway receptor ligands. Results The lncRNA sequencing analysis showed a total of 9158 differentially expressed lncRNAs. Gene ontology (GO) functional classification annotations revealed that the differentially expressed mRNA was mainly involved in immune inflammatory response, cellular transcriptional regulation and so on. Pathway analysis proved that differentially expressed mRNA was significantly related to the genes involved in rheumatoid arthritis, cellular senescence, and Notch signaling pathways. According to cis and trans prediction, Jagged1-2, Delta1-2 and Notch1-2 might be closely related to RA. MALAT1 in the RA group was lower than that in the NC group, and hsa-miR155-3p expression was significantly higher than that in the NC group. The expression of Notch pathway ligands Delta1, Delta2 Jagged1, Jagged2 and the receptors Notch1 and Notch2 in the RA group increased. Correlation analysis showed that hsa-miR155-3p was inversely proportional to MALAT1, hsa-miR155-3p was directly proportional to Notch pathway Delta1, Jagged1, and MALAT1 was inversely proportional to Jagged2. Conclusion In patients with rheumatoid arthritis, lncRNA MALAT1 is reduced and hsa-mir155-3p is raised, which jointly regulate the change of Notch signaling pathway.

"Efficacy and safety of Xinfeng capsule in the treatment of osteoarthritis: a multicenter, randomized, double-blinded, controlled trial".

OBJECTIVE: To evaluate the efficacy and safety of Xinfeng capsule (XFC) in patients with osteoarthritis (OA). METHODS: This was a multicenter, double-blinded, randomized, controlled, clinical trial. Patients with OA were assigned to the XFC group [treated with XFC and a glucosamine (GS) placebo, n = 129] or the GS group (treated with GS and an XFC placebo, n = 126). Both groups were treated for 4 weeks. The primary endpoint was the difference between the two groups in the Western Ontario and McMaster Universities OA (WOMAC) index total score at 4th week. The secondary endpoints were the visual analogue scale for pain, Lequesne index, function influence index rating, quality of life as assessed by the Short Form-36, erythrocyte sedimentation rate, and C-reactive protein concentration at baseline and at second week and 4th week. Bone mineral density were checked by X ray absorptiometry at baseline and 4th week. RESULTS: After 4 weeks of treatment, all patients in both groups showed similar significant improvements compared with baseline. There were no significant differences between groups regarding pain relief, bilateral femoral bone mineral density, and laboratory indices such as erythrocyte sedimentation rate and C-reactive protein concentration. Both groups had a significantly lower function influence index rating score and curative effect for each sign/symptom in week 4 than in week 0, and these changes did not significantly differ between groups. XFC was superior to GS in improving the WOMAC index total score, WOMAC scores for function and stiffness, integrated symptoms, physiological function, energy, emotional function, mental health, and health?changes. Fourteen adverse reactions were reported, and the incidence of adverse reactions did not significantly differ between groups. The most common adverse reactions were hepatic impairment, kidney functional damage, gastrectasia, and facial skin allergy. The types of adverse reactions did not differ between groups. CONCLUSION: XFC is effective and safe in the treatment of OA. XFC was superior to GS in improving the WOMAC index total score, WOMAC scores for pain, stiffness, and function, visual analogue scale for pain, Lequesne index, and Short Form-36 quality of life.

[Effect of Triptolide on Cardiac Function in Arthritic Rats and Its Mechanism].

OBJECTIVE: To observe the changes of cardiac function in arthritic rats and the effect of triptolide on it. METHODS: Forty rats were divided in random into normal control (NC) group, model control (MC) group, leflunomide (LEF) group and triptolide (TP) group. Except for the normal group, rats in the other three groups were injected with Freund's complete adjuvant to create arthritic inflammation in the right hind paws, and the interventional drug was administered on the 12th day after the inflammation. By treating for 30 d, the cardiac function of rats was detected by left ventricular catheterization. The expressions of superoxide dismutase (SOD), malondialdehyde (MDA), reacitve oxygen species (ROS), total antioxidation (T-AOC), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) in serum were measured by enzyme-linked immunosorbent assay. The expressions of keap-like protein 1 ( Keap1), muscular aponeurotic fibrosarcom ( maf) and nuclear factor-E2 related factor2 ( Nrf2) mRNAs in cardiac tissue were detected by real-time PCR. The expressions of Keap1, maf and Nrf2 proteins in heart tissues were detected by Western blot. RESULTS: Comparing with the normal group, the heart rate (HR), heart index (HI), left ventricular systolic pressure (LVSP), and left ventricular end-diastolic pressure (LVEDP) of the model group were significantly increased, whereas the maximum change rate of ventricular pressure rise or decline (±dp/dtmax) was significantly decreased ( P<0.01). SOD, MDA, ROS, T-AOC, and TNF-α were all increased, and IL-10 was significantly decreased ( P<0.01). The mRNA and protein expressions of Keap1, maf and Nrf2 in heart tissues were increased ( P<0.01). Comparing with the model group, HR, HI, LVSP, and LVEDP in the triptolide group were significantly decreased, whereas the ±dp/dtmax was significantly increased ( P<0.01). SOD, MDA, T-AOC, ROS, TNF-α decreased while the IL-10 increased ( P<0.05, P<0.01). The expressions of Keap1, maf and Nrf2 mRNAs and proteins in the heart tissues of the triptolide group were decreased ( P<0.01). CONCLUSION: Triptolide could improve cardiac function in arthritic rats, and the mechanism may related to its ability of improving the anti-oxidationin cardiomyocytes, reducing oxidative stress damage, and inhibiting abnormal immune inflammatory response.

A Novel Chinese Medicine, Xinfeng Capsule, Modulates Proinflammatory Cytokines via Regulating the Toll-Like Receptor 4 (TLR4)/Mitogen-Activated Protein Kinase (MAPK)/Nuclear Kappa B (NF-κB) Signaling Pathway in an Adjuvant Arthritis Rat Model.

BACKGROUND Rheumatoid arthritis (RA) is a chronic autoimmune disease targeting joints. This research aimed to explore the effects of Xinfeng capsules (XFC) on cardiac injury in adjuvant arthritis (AA) model rats and assessed the associated mechanism. MATERIAL AND METHODS An adjuvant arthritis (AA) rat model was established by intracutaneously injection with Freund's complete adjuvant (FCA). Model rats were divided into 4 groups: an AA model group, an astragalus polysaccharides (APS) group, a methotrexate (MTX) group, and an XFC and triptolide (TPT) group. Hematoxylin-eosin (HE) staining was used to observe histopathologic changes. TUNEL assay was utilized to evaluate the apoptosis of cardiomyocytes. ELISA was utilized to evaluate levels of tumor necrosis factor alpha (TNF-alpha), interleukin 17 (IL-17), and interleukin 6 (IL-6) in myocardial tissues. Quantitative RT-PCR (qRT-PCR) was used to detect microRNA-21 (miRNA21) levels. Mitogen-activated protein kinase (MAPK)/p38, Toll-like receptor 4 (TLR4), and nuclear kappa B (NF-kappaB)/p65 levels were evaluated using Western blot. RESULTS XFC significantly improved proinflammatory response compared to the AA model group (p<0.05). XFC treatment significantly decreased the number of cells staining TUNEL-positive compared with the model group (p<0.05). XFC treatment significantly reduced TNF-alpha, IL-17, and IL-6 levels in myocardial tissues compared to the model group (p<0.05). Levels of miRNA21 were significantly lower in the XFC group compared to the AA model group (p<0.05). TLR4, MAPK/p38, and NF-kappaB/p65 expression levels were significantly lower in the XFC group than in the model group (p<0.05). CONCLUSIONS Xinfeng capsule, a traditional Chinese medicine preparation, protects against cardiac injury in AA rats by modulating proinflammatory cytokines expression via the TLR4/MAPK/NF-kappaB signaling pathway.