FTO-mediated RNA m6A methylation regulates synovial aggression and inflammation in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) plays an important role in synovial inflammation and joint damage in rheumatoid arthritis (RA). As the most abundant mRNA modification, N6-methyladenosine (m6A) is involved in the development of various diseases; however, its role in RA remains to be defined. In this study, we reported the elevated expression of the m6A demethylase fat mass and obesity-associated protein (FTO) in FLS and synovium from RA patients. Functionally, FTO knockdown or treatment with FB23-2, an inhibitor of the mRNA m6A demethylase FTO, inhibited the migration, invasion and inflammatory response of RA FLS, however, FTO-overexpressed RA FLS exhibited increased migration, invasion and inflammatory response. We further demonstrated that FTO promoted ADAMTS15 mRNA stability in an m6A-IGF2BP1 dependent manner. Notably, the severity of arthritis was significantly reduced in CIA mice with FB23-2 administration or CIA rats with intra-articular injection of FTO shRNA. Our results illustrate the contribution of FTO-mediated m6A modification to joint damage and inflammation in RA and suggest that FTO might be a potential therapeutic target in RA.

NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis.

OBJECTIVE: Recent studies indicate that N-acetyltransferase 10 (NAT10)-mediated ac4C modification plays unique roles in tumour metastasis and immune infiltration. This study aimed to uncover the role of NAT10-mediated ac4C in fibroblast-like synoviocytes (FLSs) functions and synovial immune cell infiltration in rheumatoid arthritis (RA). METHODS: FLSs were obtained from active established patients with RA. Protein expression was determined by western blotting or immunohistochemistry or multiplexed immunohistochemistry. Cell migration was measured using a Boyden chamber. ac4C-RIP-seq combined with RNA-seq was performed to identify potential targets of NAT10. RNA immunoprecipitation was used to validate the interaction between protein and mRNA. NAT10 haploinsufficiency, inhibitor remodelin or intra-articular Adv-NAT10 was used to suppress arthritis in mice with delayed-type hypersensitivity arthritis (DYHA) and collagen II-induced arthritis (CIA) and rats with CIA. RESULTS: We found elevated levels of NAT10 and ac4C in FLSs and synovium from patients with RA. NAT10 knockdown or specific inhibitor treatment reduced the migration and invasion of RA FLSs. Increased NAT10 level in the synovium was positively correlated with synovial infiltration of multiple types of immune cells. NAT10 inhibition in vivo attenuated the severity of arthritis in mice with CIA and DTHA, and rats with CIA. Mechanistically, we explored that NAT10 regulated RA FLS functions by promoting stability and translation efficiency of N4-acetylated PTX3 mRNA. PTX3 also regulated RA FLS aggression and is associated with synovial immune cell infiltration. CONCLUSION: Our findings uncover the important roles of NAT10-mediated ac4C modification in promoting rheumatoid synovial aggression and inflammation, indicating that NAT10 may be a potential target for the treatment of RA, even other dysregulated FLSs-associated disorders.

Coptisine inhibits aggressive and proliferative actions of fibroblast like synoviocytes and exerts a therapeutic potential for rheumatoid arthritis.

OBJECTIVE: Coptisine, a natural bioactive small molecular compound extracted from traditional Chinese herb Coptis chinensis, has been shown to exhibit anti-tumor effect. However, its contribution to autoimmune diseases such as rheumatoid arthritis (RA) is unknown. Here, we evaluate the effect of coptisine in controlling fibroblast-like synoviocytes (FLS)-mediated synovial proliferation and aggression in RA and further explore its underlying mechanism(s). METHODS: FLS were separated from synovial tissues obtained from patients with RA. Protein expression was measured by Western blot or immunohistochemistry. Gene expression was detected by quantitative RT-PCR. The EdU incorporation was used to measure cell proliferation. Migration and invasion were determined by Boyden chamber assay. RNA sequencing analysis was used to seek for the target of coptisine. The in vivo effect of coptisine was evaluated in collagen-induced arthritis (CIA) model. RESULTS: Treatment with coptisine reduced the proliferation, migration, and invasion, but not apoptosis of RA FLS. Mechanistically, we identified PSAT1, an enzyme that catalyzes serine/one-carbon/glycine biosynthesis, as a novel targeting gene of coptisine in RA FLS. PSAT1 expression was increased in FLS and synovial tissues from patients with RA compared to healthy control subjects. Coptisine treatment or PSAT1 knockdown reduced the TNF-α-induced phosphorylation of p38, ERK1/2, and JNK MAPK pathway. Interestingly, coptisine administration improved the severity of arthritis and reduced synovial PSAT1 expression in mice with CIA. CONCLUSIONS: Our data demonstrate that coptisine treatment suppresses aggressive and proliferative actions of RA FLS by targeting PSAT1 and sequential inhibition of phosphorylated p38, ERK1/2, and JNK MAPK pathway. Our findings suggest that coptisine might control FLS-mediated rheumatoid synovial proliferation and aggression, and be a novel potential agent for RA treatment.

Response to belimumab among patients with refractory lupus nephritis: a real-world observational retrospective multicenter study.

OBJECTIVES: Belimumab is a biological agent approved for the treatment of active lupus nephritis (LN), but its efficacy on refractory lupus nephritis (LN) is unknown. This study aims to evaluate the efficacy and safety of belimumab in Chinese patients with refractory LN. METHODS: This multicenter, observational, and retrospective study enrolled patients with refractory LN who failed induction therapy with steroids, cyclophosphamide, mycophenolate, and calcineurin inhibitors and received 24-week belimumab treatment before data analysis. Treatment outcomes include the overall clinical response (physician judgment, disease activity, organ damage) and renal response (complete renal response, partial renal response, no renal response). Laboratory indices and adverse events were recorded as well. RESULTS: Of the 45 patients enrolled in the study, 6 (13.3%) achieved complete renal response, 19 (42.2%) achieved partial renal response, and the overall renal response rate was 55.6%. Median rSLEDAI decreased from 12 (IQR 8-12) at baseline to 8 (IQR 4-8) (p < 0.0001), 4 (IQR 4-8) (p < 0.0001) at 12 and 24 weeks. Mean urinary protein decreased more than 50% from 3.2 g/24 h at baseline to 1.0 g/24 h at 24 weeks (p < 0.0001). The conditions of hypoalbuminemia and hypocomplementemia had also gradually improved. The levels of autoantibodies showed a significant downward trend. Additionally, 9 (20.0%) patients successfully reduced the dosage of prednisone to a safe range, and 3 of them achieved their treatment goal of prednisone cessation. The mean prednisone dosage decreased from 32.7 mg/day at baseline to 18.6 mg/day (p < 0.0001), 13.3 mg/day (p < 0.0001) at 12 and 24 weeks. There were 3 adverse events reported, including 2 cases of infection, and 1 case of allergy. No serious events occurred during the follow-up. CONCLUSIONS: Belimumab is effective and safe when used in clinical practice, which can be considered as an add-on therapy for refractory LN. Key Points • A multicenter observational study in the real clinical settings of China. • First revealed the efficacy and safety of belimumab in Chinese patients with refractory LN.

ALKBH5-Mediated RNA m6 A Methylation Regulates the Migration, Invasion, and Proliferation of Rheumatoid Fibroblast-Like Synoviocytes.

OBJECTIVE: Fibroblast-like synoviocytes (FLSs) are critical for promoting joint damage in rheumatoid arthritis (RA). N6 -methyladenosine (m6 A) modification plays key roles in various diseases, but its role in the pathogenesis of RA is largely unknown. Here, we investigate increased demethylase ALKBH5 promotion of proliferation, migration, and invasion of RA FLSs via regulating JARID2 expression. METHODS: ALKBH5 expression in FLSs was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. 5-ethynyl-2'-deoxyuridine, scratch wound healing, and transwell assays were implemented to determine the role of ALKBH5 on RA FLS proliferation, mobility, and migration. Then, m6 A sequencing combined with RNA sequencing was performed to identify the potential targets of ALKBH5. RNA immunoprecipitation and RNA pulldown were then used to validate the interaction between the protein and messenger RNA (mRNA). Collagen-induced arthritis (CIA) and delayed-type hypersensitivity arthritis (DTHA) models were further established to assess the therapeutic potency of ALKBH5 in vivo. RESULTS: We demonstrated that ALKBH5 expression was increased in FLSs and synovium from RA. Functionally, ALKBH5 knockdown inhibited the proliferation, migration, and invasion of RA FLSs, whereas overexpression of ALKBH5 displayed the opposite effect. Mechanistically, ALKBH5 mediated m6 A modification in the JARID2 mRNA and enhanced its mRNA stability in cooperation with IGF2BP3. Intriguingly, the severity of arthritis was attenuated in mice with DTHA and ALKBH5 knockout or rats with CIA and intra-articular injection of ALKBH5 short hairpin RNA. CONCLUSION: Our findings suggest that ALKBH5-mediated m6 A modification is crucial for synovial hyperplasia and invasion in RA. ALKBH5 might be a potential therapeutic target for RA and even for dysregulated fibroblasts in a wide range of diseases.

Schisandrin treatment suppresses the proliferation, migration, invasion, and inflammatory responses of fibroblast-like synoviocytes from rheumatoid arthritis patients and attenuates synovial inflammation and joint destruction in CIA mice.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease causing joint dysfunction. As disease-modifying anti-rheumatic drugs (DMARDs) have poor efficacy in 20% to 25% of RA patients, additional novel RA medications are urgently needed. Schisandrin (SCH) has multiple therapeutic effects. However, whether SCH is effective against RA remains unknown. PURPOSE: To investigate how SCH affects the abnormal behaviours of RA fibroblast-like synoviocytes (FLSs) and further elucidate the underlying mechanism of SCH in RA FLSs and collagen-induced arthritis (CIA) mice. METHODS: Cell Counting Kit-8 (CCK8) assays were used to characterize cell viability. EdU assays were performed to assess cell proliferation. Annexin V-APC/PI assays were used to determine apoptosis. Transwell chamber assays were used to measure cell migration and invasion in vitro. RT-qPCR was used to assess proinflammatory cytokine and MMP mRNA expression. Western blotting was used to detect protein expression. RNA sequencing was performed to explore the potential downstream targets of SCH. CIA model mice were used to assess the treatment efficacy of SCH in vivo. RESULTS: Treatments with SCH (50, 100, and 200 µΜ) inhibited RA FLSs proliferation, migration, invasion, and TNF-α-induced IL-6, IL-8, and CCL2 expression in a dose-dependent manner but did not affect RA FLSs viability or apoptosis. RNA sequencing and Reactome enrichment analysis indicated that SREBF1 might be the downstream target in SCH treatment. Furthermore, knockdown of SREBF1 exerted effects similar to those of SCH in inhibiting RA FLSs proliferation, migration, invasion, and TNF-α-induced expression of IL-6, IL-8, and CCL2. Both SCH treatment and SREBF1 knockdown decreased activation of the PI3K/AKT and NF-κB signalling pathways. Moreover, SCH ameliorated joint inflammation and cartilage and bone destruction in CIA model mice. CONCLUSION: SCH controls the pathogenic behaviours of RA FLSs by targeting SREBF1-mediated activation of the PI3K/AKT and NF-κB signalling pathways. Our data suggest that SCH inhibits FLS-mediated synovial inflammation and joint damage and that SCH might have therapeutic potential for RA.

Myricitrin inhibits fibroblast-like synoviocyte-mediated rheumatoid synovial inflammation and joint destruction by targeting AIM2.

Objective: To explore the effect and underlying mechanism of Myricitrin (Myr) in regulating fibroblast-like synoviocyte (FLS)-mediated synovitis and joint destruction in RA. Methods: FLSs were isolated from synovial tissues from patients with RA. Gene expression was measured using quantitative RT-qPCR. Protein expression was detected by immunohistochemistry or Western blot. Cell apoptosis was performed by an Annexin-PI staining assay. EdU incorporation was used to assess the proliferation of RA FLS. Transwell assay was used to characterize the cell migration and invasion ability of RA FLS. The potential target of Myr was identified by RNA sequencing analysis. The in vivo effect of Myr was assessed in a collagen-induced arthritis (CIA) model. Results: Myr treatment inhibited the lamellipodia formation, migration, and invasion, but not the apoptosis and proliferation, of RA FLSs. Myr also reduced the expression of CCL2, IL-6, IL-8, MMP-1, MMP-3, and MMP-13 induced by TNF-α. The RNA-seq results indicated that AIM2 may be a target gene of Myr in RA FLSs. Furthermore, compared to healthy controls, AIM2 expression showed higher levels in synovial tissues and FLSs from RA patients. AIM2 knockdown also inhibited RA FLS migration, invasion, cytokine, and MMP expression. In addition, either Myr treatment or AIM2 knockdown reduced the phosphorylation of AKT induced by TNF-α stimulation. Importantly, Myr administration relieved arthritis symptoms and inhibited AIM2 expression in the synovium of CIA mice. Conclusion: Our results indicate that Myr exerts an anti-inflammatory and anti-invasion effect in RA FLSs and provide evidence of the therapeutic potential of Myr for RA.

Raman spectroscopy and mapping technique for the identification of expired drugs.

As expired medical products can be repackaged and sold by unscrupulous counterfeiters, it is essential to find a rapid and convenient method for distinguishing expired and unexpired drugs. Standard detection methods such as high-performance liquid chromatography (HPLC) and thin-layer chromatography are complex, time-consuming, and require organic solvents (that are environmentally unfriendly). Additionally, the Pharmacopoeia publications do not include information about identifying expired drugs. In this study, we proposed a novel method for identifying expired medications based on Raman spectra and verified it using >20 types of expired (Old) and unexpired (New) drugs, each type from the same manufacturer. A portable Raman spectrometer was used to collect Raman spectra of all samples and the similarities between the Old and New drugs (SN-O) were evaluated. Drugs with SN-O values <0.9 were classified directly as expired drugs. For drugs with SN-O values >0.9, the content of active pharmaceutical ingredient (API) might be so low (below or around 10 wt%) that its Raman signal is largely obscured by that of the excipients. In such cases, changes in the API content are undetectable using the portable instrument. Therefore, we adopted Raman mapping technology and established a virtual imaging map to locate areas of high API content. The similarities between the Old or New spectrum and that of the API (SO-A and SN-A, respectively) were calculated after removing the signal from the excipients. Our novel methods provide a precise, rapid, convenient, and environmentally friendly way to identify expired drugs that is more effective than the standard HPLC assay.

Health-Related Quality of Life of Medical Students in a Chinese University: A Cross-Sectional Study.

Thus far, there have been no studies adapting the Mandarin 36-Item Short Form Health Survey (the SF-36) questionnaire for assessment of the health-related quality of life (HRQOL) of medical students in China. This study aimed to explore the feasibility of that form and analyse its impact factors. The study involved 498 randomly sampled medical students stratified by their academic majors, and general information was collected. The effective response rate was 83.53%. Split-half reliability coefficients and Cronbach's α coefficients of seven dimensions were more than 0.7 with the exception of the social function (SF) dimension. Spearman's correlation analysis results were basically in accord with the theoretical construction of the SF-36. The HRQOL of the students was scored from 43.83 (the RE dimension) to 93.34 (the PF dimension). The primary impact factors affecting the HRQOL of medical students included major, sleep quality, degree of physical exercise, post-exercise status, relationship with roommate, and satisfaction with family. These findings suggested that the Mandarin SF-36 was reliable for measuring the HRQOL, that the HRQOL of medical students in a Chinese university was relatively poor, and that its improvement requires concerted efforts.

Bioassay-guided isolation of antioxidant and α-glucosidase inhibitory constituents from stem of Vigna angularis.

Stem of Vigna angularis (Willd.) Ohwi & H. Ohashi (VAS) is a main byproduct with considerable bioactivities. In present study, a bioassay-guided phytochemical investigation was used and led to the isolation of 16 compounds including one new compound (1) and one compound (2) isolated from nature source firstly along with 14 known compounds (3-16). The structures of isolates were identified by NMR and HR-ESI-MS data. The ability of antioxidant and α-glucosidase inhibition of the compounds were measured in vitro. Most of the ingredients shown strong ABTS radical scavenging activity (IC50 = 4.21-14.93 µM) and α-glucosidase inhibitory activity (IC50 = 0.05-34.14 µM). Enzyme kinetic analysis and molecular docking of compounds 1 and 2 were conducted. Compounds 1 and 2 were competitive inhibitor for α-glucosidase, with the inhibition kinetic constant value of 1.03 and 1.06 µM, respectively. The potent α-glucosidase inhibitory ability of compounds 1 and 2 resulted from firm binding with the active site of α-glucosidase.

Correlation between major adverse cardiac events and coronary plaque characteristics.

BACKGROUND: Unstable plaque is believed to be responsible for major adverse cardiac events (MACE). OBJECTIVE: To determine whether coronary computed tomography angiography (CCTA) could be used to predict future MACE. METHODS: Patients undergoing CCTA between January 2008 and February 2010 were consecutively enrolled in the study. The hospital database was screened for patients who later developed acute ST segment elevation myocardial infarction (STEMI), non-STEMI (NSTEMI) or cardiac death. Plaque scores were calculated and analyzed using one-way ANOVA to examine the relationship between plaque scores and MACE. RESULTS: Of the 8557 patients who underwent CCTA, 1055 had hospital records available for follow-up. During follow-up, 25 patients experienced MACE including death (six patients), heart failure (two patients), STEMI (11 patients) and NSTEMI (six patients). The plaque scores were significantly increased in patients who later died, developed heart failure or experienced STEMI (P<0.05). Calcification, erosion and severe stenosis were responsible for the events (P<0.05). Mild and moderate lesions, positive remodelling, drug-eluting stent placement, occlusion and diffuse lesions were not predictive of MACE (P>0.05). CONCLUSION: Severe calcification, erosion and severe stenosis predict death, heart failure and STEMI.

Comparison of coronary angiography-assisted and computed coronary tomography angiography-assisted recanalisation of coronary chronic total occlusion.

BACKGROUND: Computed coronary tomography angiography (CCTA) provides an alternative to coronary angiography (CAG) and a complementary way of imaging. OBJECTIVE: To determine whether CT assistance might help increase the recanalisation rate of coronary chronic total occlusion (CTO). METHOD: Two experienced physicians took part in the study-one specialised in both CCTA and percutaneous coronary intervention (PCI), and the other had PCI experience only and no knowledge of CCTA. Consecutive patients were enrolled if CTO was diagnosed by CAG or by CCTA. The images were analysed on a dedicated work station which examined the length and characteristics of the occlusion, the calibre of the artery, the best projection for precision guidewire penetration, the use of a side branch and calcification for landmarking and selection of most suitable guidewires. Patients underwent CAG-guided PCI or CCTA-assisted PCI. The main end point was the recanalisation rate. Secondary end points included the time for successful passage of the guidewire, fluoroscopy time, and contrast, guidewire and stent consumption. RESULTS: Thirty-six patients underwent CAG and 44 CCTA. The clinical characteristics and laboratory data of the two groups were similar (p>0.05). The patients in the CCTA group had more complex disease than those in the CAG group as shown by the J-CTO score (Multicenter CTO Registry of Japan) (p<0.05). Recanalisation was possible in 75.8% of the CAG group and 72.1% of the CCTA group. However, no statistical significance was found, p>0.05. In five of seven patients who had undergone unsuccessful PCI previously the procedure was successful at the second attempt when CCTA-assisted PCI was used. The patients were divided into those for whom the procedure was a failure or a success. The J-CTO score was an independent predictor of failure (OR=0.290, 95% CI 0.158 to 0.533). CONCLUSION: CTO with favourable characteristics does not need CCTA guidance, but CCTA can be used to recanalise CTO with unfavourable characteristics when the procedure has previously failed. ACTRN12611000368932.

Application of a new three-dimensional method for the measurement of coronary stent angulation and comparison with conventional methods.

The precision of the measurement of the angulation of coronary stents or lesions using coronary angiography (CA) and computed tomographic angiography (CTA) has not been established, and obtaining a rotating artery tree to measure angulation based on CTA is time-consuming. The aim of this study was to evaluate the utility of a new three-dimensional centerline method that we have developed for the measurement of coronary stent angulation based on CTA and to compare it with other conventional methods. We used the centerline method compacted by means of our new software, the conventional artery rotation method based on CTA and the simple CA method to measure the angulations of phantoms in vitro and stents implanted in patients. The precision and repetition of this new method was compared with those of the other two methods. The angulation values obtained from both the centerline and artery rotation methods based on CTA had high correlation and agreement with the true angulation values measured using a phantom; the 95 % confidence intervals (CIs) for the differences were -0.67° to 0.91° and -0.59° to 2.93°, respectively, while the difference between the value determined using the CA method and the true angulation of the phantoms ranged from 3° to 21.8° (median 8.1°). In clinical coronary stent measurement, the difference between artery rotation and centerline measurement was small (95 % CI -9.0° to 7.6°), and both methods had good repeatability. The time required to complete the measurement was considerably shorter (p < 0.001) using the centerline method than artery rotation method (12.5 ± 1.86 vs. 71.8 ± 13.6 s), while the CA method had poor precision and repeatability in the measurement of clinical stent angulation relative to the methods based on CTA (95 % CI -14.7° to 21.7°). Our three-dimensional centerline method based on CTA for the measurement of angulation was reliable and easy to implement in both clinical and basic research image analysis, and the centerline and conventional artery rotation methods can be used interchangeably. In addition, the value obtained for the coronary stent angulation using the CA method had a large bias.

[Coronary plaques identified by coronary computed tomography angiography and the risk factors for major adverse cardiac events: a correlation analysis].

OBJECTIVE: To investigate the correlation between the findings by coronary computed tomography angiography (CCTA) and the risk factors for major adverse cardiac events (MACE). METHODS: This cohort study involved 706 out-patients who received examination with CCTA between June, 2008 and April, 2011. The severity of coronary artery disease (CAD) was graded to normal, mild, moderate, severe, and revascularization. Pearson correlation analysis and ANOVA were used to evaluate the relationship between the risk factors for CAD and coronary plaques identified by CCTA, and the predictive accuracy was determined by the receiver-operating characteristic (ROC) curve. RESULTS: Of the 706 patients, 58.63% were found to have abnormal CCTA findings. A older age, hypertension, hyperlipidemia, diabetes mellitus, cerebral infarction, CAD, and myocardial infarction were associated with an significantly increased incidence of coronary plaques (P<0.01). The Framingham score, LDL, HCY, IMT, HDL and TC were also significantly correlated with the severity of the coronary plaques (P<0.05). The ROC curves showed that Framingham risk score (0.845), Cr (0.766), HCY (0.697), IMT (0.693) and HDL (0.316) had greater predictive value for the occurrence of coronary plaques (P<0.001). CONCLUSION: The Framingham risk score, Cr, HCY, IMT and HDL are validated by CCTA as the major coronary risk factors and can be used for screening of CAD.