OBJECTIVE:To explore the efficacy and safety of Juan Bi Pill (JBP) in treatment of active rheumatoid arthritis (RA). METHODS:From February 2017 to May 2018, 115 participants from 4 centers were randomly divided into JBP group (57 cases) and placebo group (58 cases) in a 1:1 ratio using a random number table method. Participants received a dose of JBP (4 g, twice a day, orally) combined with methotrexate (MTX, 10 mg per week) or placebo (4 g, twice a day, orally) combined with MTX for 12 weeks. Participants were required with follow-up visits at 24 and 48 weeks, attending 7 assessment visits. Participants were undergo disease activity assessment 7 times (at baseline and 2, 4, 8, 12, 24, 48 weeks) and safety assessments 6 times (at baseline and 4, 8, 12, 24, 48 weeks). The primary endpoint was 28-joint Disease Activity Score (DAS28-ESR and DAS28-CRP). The secondary endpoints included American College of Rheumatology (ACR) criteria for 20% and 50% improvement (ACR20/50), Health Assessment Questionnaire Disability Index (HAQ-DI), clinical disease activity index (CDAI), visual analog scale (VAS), Short Form-36 (SF-36) score, Medial Outcomes Study (MOS) sleep scale score, serum erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), tender joint count, swollen joint count, and morning stiffness. The adverse reactions were observed during the treatment. RESULTS:After 12 weeks of treatment, DAS28-ESR and DAS28-CRP scores in both groups were lower than before treatment (both P<0.01), while the remission rate of DAS28-ESR and DAS28-CRP and low disease activity of JBP group were higher than those in the placebo group (both P<0.01). JBP demonstrated better efficacy on ACR20 and ACR50 compliance rate at 12 and 48 weeks comparing to placebo (all P<0.05). The CDAI and HAQ-DI score, pain VAS and global VAS change of RA patients and physicians, the serum ESR and CRP levels, and the number of tenderness and swelling joints were lower than before treatment at 4, 8, 12, 24, 48 weeks in both groups (P<0.05 or P<0.01), while the reduction of above indices in the JBP group was more obvious than those in the placebo group at 12 weeks (ESR and CRP, both P<0.05) or at 12 and 48 weeks (all P<0.01). There was no difference in adverse reactions between the 2 groups during treatment (P=0.75). CONCLUSION:JBP combined with MTX could effectively reduce disease activity in patients with RA in active stage, reduce the symptoms of arthritis, and improve the quality of life, while ensuring safety, reliability, and fewer adverse effects. (Trial Registration: ClinicalTrials.gov, No. NCT02885597).

Rheumatoid arthritis (RA) is an incurable systemic autoimmune disease. Disease progression leads to joint deformity and associated loss of function, which significantly impacts the quality of life for sufferers and adds to losses in the labor force. In the past few decades, RA has attracted increased attention from researchers, the abnormal signaling pathways in RA are a very important research field in the diagnosis and treatment of RA, which provides important evidence for understanding this complex disease and developing novel RA-linked intervention targets. The current review intends to provide a comprehensive overview of RA, including a general introduction to the disease, historical events, epidemiology, risk factors, and pathological process, highlight the primary research progress of the disease and various signaling pathways and molecular mechanisms, including genetic factors, epigenetic factors, summarize the most recent developments in identifying novel signaling pathways in RA and new inhibitors for treating RA. therapeutic interventions including approved drugs, clinical drugs, pre-clinical drugs, and cutting-edge therapeutic technologies. These developments will hopefully drive progress in new strategically targeted therapies and hope to provide novel ideas for RA treatment options in the future.

Abstract Objective: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by swelling and pain in the joints, which eventually lead to joint destruction. The Juan Bi pill is a Chinese medicine that has been widely used to treat RA in China for hundreds of years. This study demonstrated the effect of the Juan Bi pill on the management of active RA.Methods: Participants received a dose (4 g, twice a day p.o.) of the Juan Bi pill or placebo (tid p.o.) for 12 weeks. Participants were required to take the Juan Bi pill for 3 months with follow-up visits at 6 and 12 months, attending seven assessment visits. Participants will undergo disease activity and safety assessments seven times (at baseline and at 1, 2, 3, 6, and 12 months). The primary endpoint was the 28-joint Disease Activity Score (DAS28-ESR and DAS28-CRP). The secondary endpoints included blood sampling for erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), Health Assessment Questionnaire Disability Index, clinical disease activity index (CDAI), visual analog scale, Short Form-36 score, and morning stiffness.Results: Altogether, 213 participants were screened, and 115 participants were randomized into two groups (Juan Bi pill group, n = 57; placebo group, n = 58); four dropped out. After 12 weeks of treatment, the DAS28-ESR and DAS28-CRP scores of the two groups were significantly reduced. The Juan Bi pill group had significantly reduced DAS28-ESR and DAS28-CRP scores and a significantly higher standard rate of the European League Against Rheumatism score than the placebo group (P < 0.05). After treatment, the number of tenderness/swelling joints, CDAI score, and serum ESR and CRP levels in the two groups demonstrated a decreasing trend, which were all reduced compared with those before treatment. In the electrocardiogram, routine blood tests, liver and kidney functions, and other indices before and after treatment, the adverse reaction rate of the two groups did not increase.Conclusions: The Juan Bi pill combined with methotrexate can effectively reduce disease activity in participants with RA in the active stage, reduce the symptoms of arthritis, and improve the quality of life, while ensuring safety, reliability, and fewer adverse effects.Trial registration: ClinicalTrials.gov, NCT02885597. Registered on 30 August 2016.

Hydrogen sulfide (H2S), an endogenous, gaseous, signaling transmitter, has been shown to have vasodilative, anti-oxidative, anti-inflammatory, and cytoprotective activities. Increasing evidence also indicates that H2S can suppress the production of inflammatory mediators by immune cells, for example, T cells and macrophages. Inflammation is closely related to an immune response in several diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and cancer. Considering these biological effects of H2S, a potential role in the treatment of immune-related RA is being exploited. In the present review, we will provide an overview of the therapeutic potential of H2S in RA treatment.

AbstractBackgroundThe methylation of lysine residues has been involved in the multiple biological and diseases processes. Recently, some particular non‐histone proteins have been elucidated to be methylated by SMYD2, a SET and MYND domain protein with lysine methyltransferase activity.MethodsSMYD2 was evaluated in synovial tissue and cells derived from rheumatoid arthritis patients. We confirmed TRAF2 could be methylated by SMYD2 using Mass spectrometry, pull‐down, immunoprecipitation, methyltransferase assay, ubiquitination assay, luciferase reporter assays, and western blot analyses. Using loss‐ and gain‐of function studies, we explored the biological functions of SMYD2 in vitro and in vivo. Using acute and chronic inflammation with different mice models to determine the impact of SMYD2.ResultsHere, we first time confirmed that the cytoplasmic protein TRAF2 as the kernel node for NF‐κB signaling pathway could be methylated by SMYD2. SMYD2‐mediated TRAF2 methylation contributed to the durative sensitization of NF‐κB signaling transduction through restraining its own proteolysis and enhancing the activity. In addition, we found knocking down of SMYD2 has different degrees of mitigation in acute and chronic inflammation mice models. Furthermore, as the lysine‐specific demethylase, LSD1 could resist methylation on TRAF2 induced by SMYD2.ConclusionsOur data uncovered an unprecedented cytoplasmic protein network that employed methylation of TRAF2 for the maintenance of NF‐κB activation during inflammatory diseases.

Dear Editor, Rheumatoid arthritis (RA) is a common chronic autoimmune disease that causes progressive joint destruction and involves severe damage to physical function and life quality.1 Without rational drug intervention, 80% patients will become incapable of mobility after 3 years of illness.2 In the pathogenesis of RA, activated fibroblast-like synoviocytes (FLS) participate in the inflammatory process of RA through their own proliferation, migration, and invasion.3, 4 However, despite the thorough research, there are still gaps in our understanding of RA pathogenesis. Therefore, it is important to better understand the etiopathogenesis of RA. Neuron navigator 2 (NAV2) belongs to the neuron navigator family which mainly regulates growth, migration, and regeneration of neurons in the nervous system and is highly expressed in the brain, kidney, liver, and also existed in the skeleton, heart, placenta, as well as abundantly elevated in colon cancers.5 Previous studies have found that NAV2 facilitates invasion of cutaneous melanoma cells and plays a vital role in the poor prognosis of melanoma patients.6 Also, in the results of proteomics, NAV2 is abnormally highly expressed in RA patients' primary FLS compared with healthy volunteers.7 However, the specific role of NAV2 in RA remains unknown. We first examined NAV2 protein level in blood samples and found a significant increase of NAV2 expression in RA patients compared with osteoarthritis (OA) patients or healthy samples (Figure 1A). The clinically relevant information and spreadsheet are shown in Table S1. Then we performed an immunofluorescence double-staining experiment. The expression of vimentin indicated that the cells derived from synovium tissues were primary FLS and NAV2 expression was significantly upregulated (Figure 1B). Moreover, we confirmed the significant elevation of NAV2 in the primary RA synovial cells by using RT-qPCR and Western blot analysis (Figure 1C). Then we observed and validated NAV2 expression in AIA rat synovial tissues. Images of rats' hind paws showed significant exacerbation in inflammation and soft tissue swelling. Micro-CT and histological analysis presented swollen joints, bone destruction, synovial membrane hyperplasia accompanying serious inflammatory cell infiltration, and joint tissues with pannus formation (Figure S1A). The model group also developed much more severity and higher incidence of arthritis as determined by arthritis scores and hind paw volumes compared to the normal group, and the mean body weights of rats in the two groups increased during the experiment (Figure S1B). We furthermore monitored the production of proinflammatory cytokines IL-Iβ, IL-6, and TNF-α in the serum by ELISA kit, as shown in Figure S2A, the levels of IL-Iβ, IL-6 and TNF-α were dramatically increased. Additionally, the protein expression of MMP-9, COX-2, and IL-6 in inflamed joints was significantly upregulated (Figure S2B), indicating that the AIA model has been successfully established. Intriguingly, expression of NAV2 markedly increased both on the protein level and mRNA level (Figure 1D). And we could see the average integrated optical density (IOD) of NAV2 protein was significantly higher in the ankle joint of AIA rats (Figure 1E). In vitro assay, first, we used MH7A cells treated with TNF-α (20 ng/ml) for 0, 3, 6, 12, 24 and 48 h to evaluate an inflammatory response (Figure S3A-G). Interestingly, NAV2 mRNA level reached a peak at 3 h and then declined, NAV2 protein level showed a continuous increase from 0 to 24 h and decreased slightly at 48 h (Figure 2A). After transfection with siRNA to knockdown NAV2 expression in MH7A cells, we could see that siRNA downregulated NAV2 mRNA and protein expression when compared with scrambled control siRNA (si Scr) (Figure 2B). Intriguingly, accompanied by a decrease of NAV2, the protein and mRNA expression of IL-6, MMP-9, and COX-2 were also decreased (Figure 2C-E). Besides, silencing NAV2 significantly decreased protein expression of ICAM-1 and VCAM-1 (Figure 2F and G). Also, the mRNA level of IL-8 declined after NAV2 knockdown (Figure 2H). More importantly, lower NAV2 expression led to the decline of IL-1β and IL-6 secretion in the supernatants of the TNF-α-stimulated MH7A cells (Figure 2I and J). Immunofluorescence double staining also displayed that the co-expressions of NAV2 and COX-2 upon TNF-α induction disappeared when NAV2 was knocked down (Figure 2K). As Wnt/β-catenin pathway was stimulated in AIA rats (Figure S2C), the protein levels of GSK-3β, β-catenin, c-myc, CyclinD1, and MMP-3 appeared to be markedly attenuated when NAV2 was silenced (Figure 2L). Furthermore, silenced NAV2 expression significantly impeded cell motility, migration and invasive capabilities. Cell growth rates in si NAV2-transfected cells were also markedly diminished (Figure 2 M). We next investigated the underlying mechanisms by which NAV2 promoted inflammatory response in RA. We found that NAV2 could be regulated by transcription factor E2F1. First, we found E2F1 increased in RA FLS and AIA rat synovial tissues (Figure 3A and B), as well as in MH7A cells subjected to TNF-α for indicated times (Figure 3C). Then overexpression of E2F1 promoted NAV2 protein level, while knockdown of E2F1 by siRNA inhibited NAV2 expression. Also, we found that overexpression of E2F1 upregulated MMP-3 and MMP-9 expression, which are the downstream proteins in Wnt/β-catenin signaling pathway and related with invasion and proliferation in RA, and also increased phenotype protein levels of VCAM-1 and ICAM-1 significantly. Whereas knockdown of E2F1 produced the opposite effect (Figure 3D-O). Immunofluorescence double analysis confirmed that treatment with si E2F1 also resulted in decreased E2F1 and NAV2 significantly (Figure 3P). Furthermore, we found that elevated E2F1 expression could enhance NAV2 transcription level dramatically by luciferase reporter assay (Figure 3Q). Then the CHIP-qPCR results indicated that E2F1 could bind to the promoter region of NAV2 from -2000 to +500 (Figure 3R and S). In conclusion, the present study revealed an important novel finding that NAV2 plays a critical role in RA, and provided a new understanding of the molecular mechanism of RA which is unique and previously unreported. We also speculate targeting NAV2 might not just affect inflammation in RA but could also interfere with a major cell-cell interaction involved in sensitization of joint-innervating neurons that drive pain in arthritis.8 Therefore, NAV2 provides an attractive novel target to intervene inflammatory diseases, especially RA. This work was supported by grants from Macau Science and Technology Development Fund (FDCT) (033/2017/AMJ, 067/2018/A2, 0007/2019/AKP, and 0052/2020/A). R. Wang, J. Mao, and Y. Zhu designed research, analyzed data, and wrote the manuscript; and R. Wang, M. Li, W. Wu, Y. Qiu, W. Hu, Z. Li, Z. Wang, Y. Yu, J. Liao, W. Su performed research. All authors read and approved the manuscript. All the experimental processes were conducted within the approved guidelines of the Ethics Review Committee for Animal Research of Macau University of Science & Technology. Appropriate measures were taken to minimize the use of animals as well as their suffering. The authors declare that they have no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

SIGNIFICANCE Inflammation and the immune response are associated with many pathological disorders including rheumatoid arthritis (RA), lupus, cancer, heart failure and cancer(s). In recent times, important roles of hydrogen sulfide (H2S) have been evidenced by researchers in inflammatory responses, as well as immunomodulatory effects in several disease models. Recent Advances: Numerous biological targets including cytochrome c oxidase, various kinases, enzymes involved in epigenetic changes, transcription factors viz. nuclear factor kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf-2), and several membrane ion channels, are shown to be sensitive to H2S and have been widely investigated in various pre-clinical models. CRITICAL ISSUES A complete understanding of the effects of H2S in inflammatory and immune response is vital in the development of novel H2S generating therapeutics. In this review, the biological effects, and pharmacological properties of H2S in inflammation and immune response will be addressed. The review will also cover some of the novel H2S releasing pro-drugs developed in recent years as tools to study this fascinating molecule. FUTURE DIRECTIONS H2S plays important roles in inflammation and immunity related processes. Future researches are needed to further assess the immunomodulatory effects of H2S and to assist in the design of more efficient H2S carrier systems, or drug formulations for the management of immune related conditions in humans.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by swelling, pain, and synovial damage. Effective methods lack in the treatment of RA. A traditional prescription in use for thousands of years in China, Huangqi Guizhi Wuwutang granule (HGWG) is still chosen to relieve pain and prevent joint malformation in RA patients. However, no evidence-based medical research has been organized to assess the effectiveness and safety of HGWG for RA. Methods/design: We will conduct a multicenter, randomized, double-blind, placebo-controlled clinical trial to determine whether HGWG can relieve pain and protect joints. We will randomly divide 120 patients with active RA into 2 groups, treated for 12 weeks. Main measurement is the rate of ACR50 score (American College of Rheumatology) from the baseline to 12 weeks. Secondary measurements include rate of ACR20/70, change of Disease Activity Score (DAS) 28, Health Assessment Questionnaire-Disability Index (HAQ-DI), Patient Assessment of Arthritis Pain, Patient Global Assessment of Arthritis, and AIS score. The time points are set as baseline, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, and 48 weeks. In addition, the rate of ACR50 from the baseline to 2 weeks, 4 weeks, 8 weeks, 24 weeks, and 48 weeks’ follow-up are also the secondary outcome measures. Discussion: The findings of this research will elucidate the efficacy and safety of HGWG and provide an alternative treatment for RA. In addition, our data will benefit the clinical decision-making on active RA and possibly be incorporated into future guidelines. Trial registration: ClinicalTrials.gov ID: NCT03593837.

Background:Rheumatoid arthritis (RA) is a chronic systemic and autoimmune inflammatory disease ending with the destruction of joints. Current therapies can relieve RA symptoms, but some also bring severe adverse events. Therefore, an effective and safe therapeutic strategy remains to be created to benefit patients with RA by large. Jia Wei Niu Bang Zi granule (NBZG) consisting of RA-fighting Chinese herbals has been used in Longhua Hospital in the last several decades. NBZG has potential therapeutic effect on RA, which should be evaluated by larger sample clinical trial.Methods:A multicenter, randomized, double-blind, placebo-controlled clinical trials will be conducted to determine the efficiency of NBZG in pain relief and joint protection. A total of 120 patients with active RA will be enrolled, and treated with NBZG or placebo for 12 weeks. The primary outcome measurements include rate of American College of Rheumatology (ACR) 50 at 12 weeks' treatment. The 2nd outcome measurements include rate change of ACR20, ACR70, the disease activity score (DAS) 28, 36-item Short-Form Health Survey Questionnaire, Health Assessment Questionnaire - Disability Index, score changes of Patient Assessment of Arthritis Pain, Patient Global Assessment of Arthritis, and the Athens insomnia scale at the same time points.Discussion:Although NBZG has shown efficacy in treating RA in Longhua Hospital for decades, the universality of this efficacy needs evaluated. The results of this trial will provide a convincing evidence about NBZG's efficacy in treating active RA in a large population.Trial registration:ClinicalTrials.gov ID: NCT03173040 (registered on May 30, 2017)

Cystathionine-γ-lyase (CSE), an enzyme associated with hydrogen sulfide (H2S) production, is an important endogenous regulator of inflammation. Jumonji domain-containing protein 3 (JMJD3) is implicated in the immune response and inflammation. Here, we investigated the potential contribution of JMJD3 to endogenous CSE-mediated inflammation in rheumatoid arthritis (RA). Upregulated CSE and JMJD3 were identified in synovial fibroblasts (SFs) from RA patients as well as in the joints of arthritic mice. Knocking down CSE augmented inflammation in IL-1β-induced SFs by increasing JMJD3 expression. In addition, CSE−/− mice with collagen-induced arthritis (CIA) developed severe joint inflammation and bone erosion. Conversely, overexpressing CSE inhibited JMJD3 expression by the transcription factor Sp-1 and was accompanied by reduced inflammation in IL-1β-treated SFs. Furthermore, JMJD3 silencing or the administration of the JMJD3 inhibitor GSK-J4 significantly decreased the inflammatory response in IL-1β-treated SFs, mainly by controlling the methylation status of H3K27me3 at the promoter of its target genes. GSK-J4 markedly attenuated the severity of arthritis in CIA mice. In conclusion, suppressing JMJD3 expression by the transcription factor Sp-1 is likely responsible for the ability of CSE to negatively modulate the inflammatory response and reduce the progression of RA.

Rheumatoid arthritis (RA) is an immune-mediated disease with the characteristics of progressive joint destruction, deformity, and disability. Epigenetic changes have been implicated in the development of some autoimmune disorders, resulting in an alteration of gene transcription. Here, we investigated how Jumonji C family of histone demethylases (JMJD3) regulated the proliferation and activation of fibroblast-like synoviocytes (FLSs), which are involved in RA joint destruction and pathologic process. The JMJD3 expression and proliferation markers in RA-FLS were higher than those in healthy-FLS and were upregulated in platelet-derived growth factor (PDGF)-induced FLS. Elevated JMJD3 promoted the proliferation and migration of FLS. Treatment with JMJD3 small interfering RNA or inhibitor glycogen synthase kinase (GSK) J4 led to decreased proliferation and migration of FLS. Interestingly, induction of proliferating cell nuclear antigen (PCNA), a major player of the cell-cycle regulation, was correlated with trimethylated lysine 27 in histone H3 loss around the gene promoters. The knockdown of JMJD3 abolished PCNA expression in PDGF-induced FLS and further inhibited cell proliferation and migration, suggesting that JMJD3/PCNA played a crucial role in aspects of FLS proliferation and migration. In vivo, the ability of GSK J4 to hinder collagen-induced arthritis (CIA) in DBA/1 mice was evaluated. We found that GSK J4 markedly attenuated the severity of arthritis in CIA mice. The therapeutic effects were associated with ameliorated joint swelling and reduced bone erosion and destruction. This study revealed how JMJD3 integrated with epigenetic processes to regulate RA-FLS proliferation and invasion. These data suggested that JMJD3 might contribute to rheumatoid synovial hyperplasia and have the potential as a novel therapeutic target for RA.-Jia, W., Wu, W., Yang, D., Xiao, C., Su, Z., Huang, Z., Li, Z., Qin, M., Huang, M., Liu, S., Long, F., Mao, J., Liu, X., Zhu, Y. Z. Histone demethylase JMJD3 regulates fibroblast-like synoviocyte-mediated proliferation and joint destruction in rheumatoid arthritis.

The purpose of the current study is to evaluate the cardioprotective effects of purified Salvia miltiorrhiza extract (PSME) on myocardial ischemia/reperfusion injury in isolated rat hearts. Hearts were excised and perfused at constant flow (7 - 9 ml.min(-1)) via the aorta. Non-recirculating perfusion with Krebs-Henseleit (KH) solution was maintained at 37 degrees C and continuously gassed with 95% O2 and 5% CO2. KH solution with or without PSME (100 mg per liter solution) was used after 30-min zero-flow ischemia for the PSME and control group, respectively. Left ventricular (LV) developed pressure; its derivatives, diastolic pressure, and so on were continuously recorded via a pressure transducer attached to a polyvinylchloride balloon that was placed in the left ventricle through an incision in the left atrium. PSME treated hearts showed significant postischemic contractile function recovery (developed pressure recovered to 44.2 +/- 4.9% versus 17.1 +/- 5.7%, P<0.05; maximum contraction recovered to 57.2 +/- 5.9% versus 15.1 +/- 6.3%, P<0.001; maximum relaxation restored to 69.3 +/- 7.3% versus 15.4 +/- 6.3%, P<0.001 in the PSME and control group, respectively). Significant elevation in end-diastolic pressure, which indicated LV stiffening in PSME hearts might have resulted from the excess high dose of PSME used. Further study will be conducted on the potential therapeutic value with lower dose of PSME on prevention of ischemic heart disease.