[Cheng's Juanbi Decoction alleviates the inflammation in collagen-induced arthritis (CIA) rats via inhibiting activation of PI3K/AKT/mTOR pathway].

Objective To investigate the potential mechanism of Cheng's Juanbi Decoction (JBT) for treating collagen-induced arthritis (CIA) in rats. Methods Female SD rats were divided into normal group, CIA model group, methotrexate (MTX) group, JBT group with different doses, and LY294002 (PI3K blocker) group. The effects of JBT on toe swelling and arthritis index of rats before and after treatment were evaluated. HE staining was used to observe the pathological changes of synovial tissues. ELISA was used to determine the levels of interleukin-1ß (IL-1ß) and tumor necrosis factor α(TNF-α) in synovium of rats. Real-time quantitative PCR was used to detect mRNA expression levels of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), beclin-1, and P62. The expressions of AKT, phosphorylated AKT (p-AKT), mTOR, phosphorylated mTOR (p-mTOR), PI3K, phosphorylated PI3K (p-PI3K), P62, beclin-1, and microtubule-associated protein 1 light chain 3B (LC3B) were detected by Western blot analysis. Results Compared with the normal group, the toe of other groups was significantly swollen 1 hour before administration. Compared with the conditions 1 hour before administration, toe swelling in the high-dose JBT group, MTX group, and LY294002 group was significantly relieved 2 hours before blood collection after 30 days of administration. JBT can significantly reduce the degree of toe swelling, arthritis index(AI) score, and the destruction of synovial tissue. The levels of IL-1ß, TNF-α, mRNA expression of PI3K, AKT, mTOR and P62, and protein levels of p-PI3K, p-AKT, p-mTOR, and P62 in synovium samples of rats in the high-dose JBT group were significantly decreased. Beclin-1 mRNA and protein expression and LC3B protein level were significantly increased. Conclusion JBT may alleviate joint inflammation by inhibiting the activation of the PI3K/AKT/mTOR signaling pathway, and the therapeutic effect of high-dose JBT is comparable to that of MTX and LY294002.

[Bioinformatics of key genes and signaling pathways in macrophages in patients with rheumatoid arthritis].

Objective To screen key genes and signaling pathways in macrophages from patients with rheumatoid arthritis(RA) by bioinformatics. Methods Download the gene chip of synovial macrophages of RA patients from the Gene Expression Omnibus (GEO) database, obtain differentially expressed genes through the GEO2R function, and use the search tool for the retrival of interacting genes/proteins (STRING) database to construct a protein-protein interaction (PPI) network. Enrichment analysis was performed on key genes in Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomics (KEGG). Results By integrating 3 gene chip datasets, 87 differentially expressed genes were obtained, and 10 key genes were further obtained. The enrichment analysis found that key genes were associated with leukocyte migration, macrophage differentiation, platelet degranulation, mitogen-activated protein kinase (MAPK) activity. Other biological processes are closely related to phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling. Conclusion Key genes of macrophages in RA patients are associated with inflammatory response and may be involved in the pathogenesis of chronic inflammation in RA.

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.

Network analysis and transcriptome profiling in peripheral blood mononuclear cells of patients with rheumatoid arthritis.

The present study aimed to investigate the differential expression of long non-coding RNAs (lncRNAs) in rheumatoid arthritis (RA). High-throughput gene sequencing technology was used to detect the expression of lncRNA and mRNA in three patients with RA (RA group) and normal controls (NC group). A Bioinformatics analysis was used to assess the effects of differentially expressed mRNAs on signaling pathways and biological functions. The selected dysregulated lncRNAs were verified by reverse transcription-quantitative (RT-q)PCR in the peripheral blood mononuclear cells (PBMCs) of patients with RA and age- and sex-matched controls. A correlation analysis was used to analyze the relationship between lncRNAs and clinical indexes. From the lncRNA sequencing data, significantly differentially expressed lncRNAs between the RA and NC groups were identified by a fold change ≥2 and P<0.05. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis suggested that the differentially expressed mRNAs were mainly involved in organelle composition, intracellular regulation, signaling pathways, cancer, virus and inflammation. A total of four of these lncRNAs were confirmed by RT-qPCR to be significantly differentially expressed (LINC00304, MIR503HG, LINC01504 and FAM95B1). Through the correlation analysis, it was confirmed that there was a strong correlation between these lncRNAs and clinical laboratory indicators and indexes such as course of disease, arthrocele and joint tenderness. Overall, the present results suggested that the expression levels of LINC00304, MIR503HG, LINC01504 and FAM95B1 in PBMCs from patients with RA may serve as potential biomarkers for RA diagnosis, influencing the occurrence and progress of RA.

[Correlations of echocardiographic parameters in Gout patients: a retrospective analysis].

OBJECTIVE: To explore the correlations of echocardiographic parameters in patients with gout. METHODS: The hospitalization data and medical records of patients with gout between January, 2012 and June, 2019 were retrieved from the database of Anhui Provincial Hospital of Traditional Chinese Medicine, and the echocardiographic parameters and clinical laboratory test results of the inflammatory, immunological and metabolic indicators were analyzed. SPSS 22.0, SPSS Clementine 11.1 Aprior and other statistical software were used to determine the association rules and carry out correlation analysis, heat map analysis and multi-factor logistic regression analysis of the indicators. RESULTS: Heat map analysis showed that the expressions of EF and SV were the most significant, followed by AODd, LADs, LVDd and FS. Cluster analysis showed that AODd, EF, FS, LADs, LVDd, and SV were all in cluster 1, and IVSTd, LVPWTd, MPAD, Pmax, and RVDd were in cluster 2. Correlation analysis showed that in the 383 patients, EF was negatively correlated with LVDd (P < 0.05) and positively correlated with FS and SV (P < 0.05); AODd was positively correlated with IVSTd, LADs, LVDd, LVPWTd, RVDd, SV, and ESR (P < 0.05); FS was positively correlated with EF and SV (P < 0.05) and negatively correlated with LVDd (P < 0.05);IVSTd was positively correlated with AODd, LADs, LVPWTd, and complement C4 (P < 0.05); LADs were positively correlated with AODd, IVSTd, MPAD, RVDd, and SV (P < 0.05); LVDd was positively correlated with AODd, IVSTd (P < 0.05), and negatively correlated with LVDd and complement C3 (P < 0.05); MPAD and LADs, HDLC and TC were positively correlated (P < 0.05)and negatively correlated with Pmax (P < 0.05); Pmax was positively correlated with LVDd, RVDd and SV (P < 0.05)and negatively correlated with FS and MPAD (P < 0.05); RVDd was positively correlated with AODd, LADs, LVDd, Pmax, SV (P < 0.05); SV was positively correlated with AODd, EF, LADs, LVDd, Pmax, and RVDd (P < 0.05); complement C3 was positively correlated with complement C4 and CRP (P < 0.05), and negatively correlated with LVPWTd (P < 0.05); complement C4 was positively correlated with IVSTd, complement C3, CRP, and ESR (P < 0.05); CRP was positively correlated with complement C3, complement C4, IgA, IgG (P < 0.05), and negatively correlated with TC, HDLC, and TG (P < 0.05); TG was positively correlated with HDLC, IgM, and TC (P < 0.05), and negatively correlated with CRP (P < 0.05); HDLC was positively correlated with MPAD, HDLC and TC (P < 0.05) and negatively correlated with CRP (P < 0.05); IgA was positively correlated with CRP, IgG and IgM (P < 0.05); IgG was positively correlated with CRP, IgA and IgM (P < 0.05); IgM is positively correlated with TG, IgA, IgG, UA (P < 0.05) and negatively correlated with CRP (P < 0.05); UA was positively correlated with IgM (P < 0.05); ESR was positively correlated with AODd and complement C4 (P < 0.05); HCY was negatively correlated with RVDd (P < 0.05); TC was positively correlated with MPAD and TG (P < 0.05), and negatively correlated with CRP (P < 0.05). The increase of Pmax was significantly associated with the increase of LDL-C, UA, complement C4, TG, HCY, HDL-C, IgG, ESR, CRP, and complement C3; the increase of SV was associated with the elevations of UA, LDL-C, complement C4, HDL-C, CRP, IgG, HCY, TC, ESR, TG, and complement C3. Multivariate logistic regression analysis indicated that FS was positively correlated with LDL-C (P < 0.05), Pmax was negatively correlated with IgM (P < 0.05), and SV was negatively correlated with ESR (P < 0.05). CONCLUSIONS: The changes of echocardiographic parameters in patients with gout are correlated with the increase in inflammation, immunity, and metabolic indexes. Patients with a history of smoking and drinking do not show obvious changes in cardiac function. The changes in metabolic indexes are risk factors for changes in echocardiographic parameters.

RNA-seq reveals the circular RNA and miRNA expression profile of peripheral blood mononuclear cells in patients with rheumatoid arthritis.

OBJECTIVE: Circular RNAs (circRNAs) are a significant class of molecules involved in a wide range of diverse biological functions that are abnormally expressed in many types of diseases. The present study aimed to determine the circRNAs specifically expressed in peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patients to identify their possible molecular mechanisms. METHODS: To identify the circRNAs specifically expressed in RA, we started by sequencing the of PBMCs circRNA and microRNAs (miRNAs) from a RA group (n = 3) and a control group (n = 3). We constructed a network of differentially expressed circRNAs and miRNAs. Then, we selected differentially expressed circRNAs in PBMCs from 10 RA patients relative to 10 age- and sex-matched controls using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Spearman's correlation test was used to evaluate the correlation of circRNAs with biochemical measurements. RESULTS: A total of 165 circRNAs and 63 miRNAs were differently expressed between RA patients and healthy people according to RNA-seq, including 109 circRNAs that were significantly up-regulated and 56 circRNAs that were down-regulated among the RA patients. RT-qPCR validation demonstrated that the expression levels of hsa_circ_0001200, hsa_circ_0001566, hsa_circ_0003972, and hsa_circ_0008360 were consistent with the results from the sequencing analysis. Then, we found that there were significant correlations between the circRNAs and disease severity. CONCLUSION: Generally, these results suggest that expression of hsa_circ_0001200, hsa_circ_0001566, hsa_circ_0003972, and hsa_circ_0008360 in PBMCs from RA patients may serve as potential biomarkers for the diagnosis of RA, and these circRNAs may influence the occurrence and development of RA.

[Differentially expressed inflammatory proteins in acute gouty arthritis based on protein chip].

OBJECTIVE: To detect the differentially expressed inflammatory proteins in acute gouty arthritis (AGA) with protein chip. METHODS: The Raybiotech cytokine antibody chip was used to screen the proteomic expression in serum samples of 10 AGA patients and 10 healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were applied to determine the biological function annotation of differentially expressed proteins and the enrichment of signal pathways. ELISA method was used to verify the differential protein expression in 60 AGA patients and 60 healthy subjects. The ROC curve was employed to evaluate the diagnostic value of differential proteins in AGA patients. RESULTS: According to|log2FC|>log2 1.2 and corrected P<0.01, 4 most differentially expressed proteins in AGA patients were identified, including tumor necrosis factor receptor super family members Ⅱ (TNF RⅡ), macrophage inflammatory protein 1ß (MIP-1ß), interleukin-8 (IL-8), and granulocyte-macrophage colony stimulating factor (GM-CSF). GO and KEGG enrichment analysis showed that the differentially expressed proteins were related to inflammation, metabolism and cytokine pathways. The ELISA results showed that serum levels of differentially expressed proteins were significantly different between AGA patients and healthy subjects(all P<0.01). ROC curve analysis showed that the areas under the curve (AUCs) of GM-CSF, IL-8, MIP-1ß and TNF RⅡ for predicting AGA were 0.657 (95% CI: 0.560-0.760, sensitivity: 68.33%, specificity: 50.00%), 0.994 (95% CI: 0.980-1.000, sensitivity: 100.00%, specificity: 61.67%), 0.980 (95% CI: 0.712-0.985, sensitivity: 95.00%, specificity: 98.33%) and 0.965 (95% CI: 0.928-1.000, sensitivity: 100.00%, specificity: 10.00%), respectively. CONCLUSIONS: Proteomics can be applied to identify the biomarkers of AGA, which may be used for risk prediction and diagnosis of AGA patients.