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.

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.

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.

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.

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.

[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.

[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.

Mechanism studies of Xinfeng capsule on improving cardiovascular function though toll-like receptor 4/nuclear factor kappa B pathway in a rat model of adjuvant arthritis.

OBJECTIVE: To observe the impact of Xinfeng capsule (XFC) on cardiovascular function in adjuvant arthritis (AA) model rats and investigate the mechanism though toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway. METHODS: Seventy rats were randomly divided into seven groups: normal control (NC), model control (MC), tripterygium glycosides tablet (TPT), methotrexate (MTX), high, moderate and low dose XFC group. The administration began from day 19 after modeling for 30 day. Paw swelling, arthritic index (AI), cardiac function indexes and myocardial pathological pattern were detected. The expression of TLR4, myeloid differentiation factor (MyD) 88, interleukin-1 receptor-associated kinase (IRAK) 1, tumor necrosis factor receptor associated factor (TRAF) 6, NF-κB, tumor necrosis factor-alpha (TNF-α) proteins in myocardial tissue were determined by western blot method. RESULTS: Paw swelling and AI in MC group increased in MC group (P < 0.01), and decreased in high and moderate dose XFC groups (P < 0.01 or P > 0.05). Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR) were elevated in MC group (P < 0.01), and ± dp/dtmax and CI were reduced (P < .01); while LVSP, LVEDP and HR declined and ±dp/ dtmax, CI improved in high dose XFC group (P < 0.05 or P < 0.01). LVSP in high dose XFC group were reduced more than other treatment groups (P < 0.05 or P < 0.01). The improvements on LVEDP, dp/ dt-max were superior to MTX and low dose XFC group, and the improvement on CI was better than low dose XFC group (P < 0.05 or P < 0.01). Myocardial fibers arranged irregular in MC group with intracellular edema and mitochondria damage. The modifications on myocardial structural were shown in each treatment group, but more prominent in TPT, high and moderate dose XFC group. The proteins of TLR4, MyD88, IRAK1, TRAF6, NF-κB, TNF-α were highly expressed in MC group, and those proteins declined in high and moderate dose XFC group (P < 0.05 or P < 0.01). High dose XFC group was superior to MTX and low dose XFC group on reducing TLR4, NF-κB, TNF-α (P < 0.05). CONCLUSION: XFC can not only inhibit the excessive activation of TLR4/NF-κB signaling pathway and the increased inflammatory mediators, but also reduce the damage of myocardial tissue and cells.

[Effect of Xinfeng Capsule on Pulmonary Function, Thi/Th2 Cells, and Regulatory T Cells of Adjuvant Arthritis Rats].

Objective To observe the effects of Xinfeng Capsule (XFC) at different doses on lung function, Thl/Th2 cells, regulatory T cells (Treg) in adjuvant arthritis (AA) rats. Methods Totally 84 rats were randomly divided into 5 groups, i.e., the normal control group (NC) , the model group (M) , the methotrexate (MTX) group, the Tripterygium Glycosides Table (TGT) group, the low dose XFC (XFC- L) group, the medium dose XFC (XFC-M) group, the high dose XFC (XFC-H) group, 12 in each group. Freund's complete adjuvant (FCA; 0. 1 mL) was intradermally injected to all rats except those in the NC group from right rear paw to induce inflammation. Medication was started from the 19th day after inflam- mation. Normal saline was administered to rats in the NC group and the M group. Rats in the rest groups were correspondingly administered with MTX, TGT, XFC, respectively. Changes of each index were ob- served in all groups. Results (1) Compared with the NC group, rat paw swelling degree (E) , arthritis index (AI) , lung index (LI) , average expiratory flow in 1 second (FEV1/FVC%) , alveolitis integral, TNF- α, Th1/Th2 cells, transforming growth factor-ß1 ( TGF-ß1 ) expression significantly increased in the M group (P <0. 01) ; forced vital capacity (FVC) , peak expiratory flow 25% of vital capacity (FEF25), peak expiratory flow 50% of vital capacity (FEF50), peak expiratory flow 75% of vital capacity (FEF75), the maximum mid-expiratory flow (MMF) , peak expiratory flow (PEF) , CD4 ⁺Treg, CD4⁺CD25 ⁺Treg, IL-10, and Foxp3 expression significantly decreased in the M group (P <0. 01). (2) Compared with the M group, body weight, FVC, FEF25, FEF50, FEF75, MMF, PEF, IL-10, Treg, and Foxp3 expression increased in all treatment groups; E, Al, LI, FEV1/FVC%, TNF-α, Th1/Th2 cells, and TGF-ß1 expression decreased in all treatment groups (P <0. 05, P <0. 01). (3) Compared with the XFC-M group, LI, alveolitis integral, TNF- α, Th1/Th2 cells, and TGF-ß1 increased; FVC, FEF25, FEF50, FEF75, IL-10, CD4⁺Treg, CD4⁺CD25⁺ Treg, and Foxp3 decreased in other treatment groups (P <0. 05, P <0. 01). Conclusions AA rats had local swollen paws and decreased lung function. XFC could significantly improve paw swelling and Al of AA rats, and improve lung function. It could reduce inflammatory reaction and immune complexes on tis- sue and organ damage, improve joint and pulmonary symptoms possibly through promoting expressions of IL-10, CD4⁺Treg, CD4⁺CD25⁺Treg, and Foxp3, and inhibiting TNF-α,Th1/Th2 cells, and TGF-ß1 ex- pression.

[Effects of Triptolide on the Autophagy in Synovial, Spleen and Thymus of Rats with Adjuvant Arthritis].

OBJECTIVE: To determine the effect of triptolide (TP) on the expression of ATG /LC3-Ⅱ Beclin1 in synovial, spleen, and thymusof rats with adjuvant arthritis (AA). METHODS: Rats were divided for four groups: normal control (NC), model control (MC), leflunomide (LEF) treatment, and triptolide (TP)treatment, with 12 rats in each group.The AA model was established through Freund's complete adjuvant (0.1 mL each) injection into the right foot plantar skin to introduce inflammation and 10 days of tail root injection of 0.05 mL Freund's complete adjuvant for immunity strengthening. Drug administration started 13 days after induction of inflammation. Rats in the NC and MC groups were given normal saline (1 mL/100 g) once a day for 30 days, compared with 5 mg/kg of oral LEF for the rats in the LEF group and 50 µg/kg of oral TP for the rats in the TP group. Paw swelling (E), joint arthritis index(AI) and joint pathological changes of the rats were recorded. The serum expressions of cytokines B lymphocyte stimulating factor (BAFF), interleukin (IL)-1, tumor necrosis factor (TNF) alpha, IL-15,and IL-10 were detected by ELISA. The expressions of Atg5, Atg7, and Atg12 mRNA in synovial, spleen, and thymus of the rats were detected by RT-PCR.The expressions of LC3-Ⅱ and Beclin1 in synovial, spleen, and thymus of the rats were detected by Western blot assay. RESULTS: The AA model rats had lower serum BAFF, IL-1, TNF alpha, IL-15, and IL-10; lower Atg5and Atg12 mRNA in synovial; lower Atg5 mRNA, Atg7, and Atg12 mRNA in spleen; higher Atg12 mRNA in thymus; and lower LC3-Ⅱ and Beclin1 in synovial, spleen and thymus(P<0.05 or 0.01). TP treatment led to reduced paw swelling and arthritis index; declined Atg7 and Atg12 mRNA in synovial; declined Atg5, Atg7 mRNA and Atg12 mRNA in spleen; decreased Atg5 and Atg7mRNA in thymus; increased Atg12 mRNA in thymus; and increased LC3-Ⅱ and Beclin1 in synovial, spleen and thymus (P<0.05 or 0.01). Compared with rats treated with LEF, TP treated rats had lower TNF-α and BAFF and higher E and IL-15 (P<0.05 or 0.01); as well as decreased expressions of Atg7 mRNA (synovial) and Atg5, Atg7 mRNA (thymus), and increased expressions of Atg12 mRNA (thymus) and Atg5, Atg7, Atg12 mRNA (spleen). CONCLUSION: TP regulates autophagy in synovial, thymus and spleen of AA rats, and improves theirjointinflammatory response.

Urinary metabolite profiling provides potential differentiation to explore the mechanisms of adjuvant-induced arthritis in rats.

To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant-induced arthritis (AA). Sprague-Dawley rats were randomly divided into a control group and an experimental group, with eight in each. Rats in the experimental group were induced by intracutaneous innoculation of 0.1 mL Freund's complete adjuvant to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC-TOF/MS technology with multivariate statistical approaches, including principal component analysis, partial least squares discriminant analysis and orthogonal projections to latent structures-discriminant analysis. Nine potential biomarkers were identified, including 2,2-dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, l-dopa, 1,4-dihydroxy-2-naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of Xinfeng capsule on nuclear factor Kappa B/tumor necrosis factor alpha and transforming growth factor beta 1/Smads pathways in rats with cardiac injuries induced by adjuvant arthritis.

OBJECTIVE: To investigate effects of Xinfeng capsule (XFC) on cardiac function in rats with adjuvant arthritis (AA) and explore the mechanism of these effects. METHODS: Forty-eight rats were randomly divided into normal control (NC), model control (MC), methotrexate (MTX) and XFC groups of equal size. In all groups except for the NC group, 0.1 mL Freund's complete adjuvant (FCA) was intracutaneously injected in the right rear vola pedis to induce inflammation. Drugs were applied beginning 19 days after induction of inflammation. Normal saline was administered to the NC and MC groups and 1 mg/ 100 g MTX (weekly) and 0.12 g/100 g XFC (daily) to the MTX and XFC groups, respectively. Rats were sacrificed after 30 day of treatment. Toe swelling degree (TSD), arthritis index (Al), cardiac function and expression of nuclear factor kappa B (NF-κB)/tumor necrosis factor alpha (TNF-α) and transforming growth factor beta 1 (TGF-ß1)/Smads pathway proteins were measured. RESULTS: In the MC group, TSD and Al were greatly increased, while parameters of cardiac function were decreased and morphological analysis showed myocardial cell damage. Expression of TNF-α, NF-KB, Smad2, P-Smad2, Smad4 and TGF-ß1 proteins were elevated in cardiac tissue, while Smad7 expression was decreased. TSD and Al values closely correlated to parameters of cardiac function and to levels of proteins in the NF-κB/TNF-α and TGF-ß1/Smads pathways. Certain correlations were identified among TGF-ß1 and NF-KB, Smad2, P-Smad2 and Smad4. With XFC intervention, both TSD and Al were decreased and parameters of cardiac function and ultrastructure of myocardial cells improved. Expressions of NF-κB, Smad2, and Smad4 proteins were greatly decreased and Smad7 expression was elevated, as compared with levels in the MC and MTX groups. CONCLUSION: XFC regulates expression of proteins in the NF-KB/TNF-α and TGF-ß1/Smads pathways, decreases immune complex deposition in cardiac tissue and improves cardiac function in AA rats via upregulation of Smad7.

Efficacy and safety of Xinfeng capsule in patients with rheumatoid arthritis: a multi-center parallel-group double-blind randomized controlled trial.

OBJECTIVE: To evaluate the efficacy. and safety of Xinfeng capsule in patients suffering rheumatoid arthritis (RA). METHODS: A multi-center parallel-group designed, double-blind, randomized, controlled trial was conducted. Totally 304 RA patients were assigned to two groups: one group was administered Xinfeng capsule (XFC) plus the placebo of leflunomide and the other given leflunomide (LEF) plus the placebo of XFC for twelve weeks. The clinical and laboratory parameters were compared at baseline and fourth, eighth, and twelfth weeks. RESULTS: After twelve-week treatment, patients in two groups all showed some trend of effectiveness when compared in terms of American Rheumatism Association (ACR) recommended 20%, 50%, 70% improvement criteria, but it was insignificant. The validity in ameliorate modified disease activity score (DAS28) and laboratory indexes as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF) were also found no difference. The score of health assessment questionnaire (HAQ), self-rating anxiety scale (SAS), self-rating depression scale (SDS) and quality of life questionnaire with rheumatoid arthritis (RAQOL) both lower than the first week and the changes showed no difference. However, the score of SDS dropped more in XFC group than in the other. A total of 147 adverse reaction cases were reported, which shows no difference between the two groups. The most common adverse reactions were hepatic impairment, anemia, leukocytopenia, epigastric discomfort and phalacrosis. CONCLUSION: XFC demonstrated better improvement in the scores of SDS and compared with those of LEF group.

[Effect of Xinfeng Capsule on Lipoprotein Metabolism of Rheumatoid Arthritis Patients].

OBJECTIVE: To explore the effect of Xinfeng Capsule (XC) on lipoprotein metabolism of rheumatoid arthritis (RA) patients. METHODS: Totally 180 RA patients were assigned to the experimental group and the control group by random digit table, 90 in each group. Patients in the experimental group took XC (three pills each time, three times daily), while those in the control group took Methotrexate Tablet (four tablets each time, once per week). One month consisted of one therapeutic course and all patients were treated for two therapeutic courses. A healthy control group consisting of 60 patients was also set up. Changes of lipoprotein indices, clinical efficacy, lipid metabolism, joint symptoms and signs, activity indicators were observed, and correlation analyses were performed. RESULTS: Compared with the healthy control group, expression levels of prealbumin (PA), globulin (GLO), high-density lipoprotein (HDL), apolipoprotein Al (Apo-A1) were lowered in RA patients (P <0. 05, P <0. 01). Correlation analyses showed that PA was negatively correlated with joint tenderness, morning stiffness time, disease activity score (DAS-28), C-reactive protein (CRP), interleukin (IL)-6, respectively. Total protein (TP) was negatively correlated with joint tenderness. GLO was negatively correlated with joint tenderness and DAS-28. HDL was negatively correlated with erythrocyte sedimentation rate (ESR) and endothelin (ET)-1. Apo-Al was negatively correlated with joint pain; Apo-B was negatively correlated with CRP; LDL was negatively correlated with morning stiffness time (P <0. 05, P <0. 01). Compared with before treatment, expression levels of PA, HDL, Apo-A1 , Apo-B, and serum IL-10 contents increased, and expression levels of ESR, CRP, IL-6, ET-1 , joint pain, joint swelling, morning stiffness time, and DAS-28 decreased in the experimental group (P <0. 05, P <0. 01). PA increased more after treatment than before treatment in the control group (P <0. 01). There was statistical difference in joint symptoms (except joint tenderness) and activity indices (except ET-1) in the control group (P <0. 05, P <0. 01). Compared with the control group after treatment, PA and HDL increased, ET-1 and duration of morning stiffness decreased in the experimental group (all P <0. 05). CONCLUSIONS: Lipoprotein metabolic disorder exists in RA patients, and it is associated with disease activity. XC could obviously improve lipoprotein metabolism and joint symptoms.

Effect of xinfeng capsule on pulmonary function in a adjuvant arthritis rat model.

OBJECTIVE: To observe the relationship between reduced pulmonary function and regulatory T cells (Tregs) and helper T cells (Th)1/Th2 drift in a rat model of adjuvant arthritis (AA), and to study the impact of Xinfeng capsule (XFC) on pulmonary function and investigate the mechanism of action. METHODS: Forty rats were randomly divided into normal control group (NC), model control group (MC), Tripterygium glycosides tablet group (TPT), and XFC group, with 10 in each. Except for the NC group, AA was induced in all rats by intracutaneous injection of 0.1 mL Freund's complete adjuvant in the right paw. On the 19th day after modeling, the NC and MC groups were given physiological saline (0.9%), while the TPT and XFC groups were given TPT (10 mg/kg) and XFC (2.4 g/kg), once daily, respectively. Thirty days after administration, changes in paw swelling, arthritis index (AI), pulmonary function, levels of serum gamma-interferon (IFN-gamma) and interleukin (IL)-4, Tregs in peripheral blood, and IFN-gamma, IL-4, Forkhead box transcription factor 3 (FoxP3) in lung tissue were observed by enzyme-linked immunosorbent assay, flow cytometry, polymerase chain reaction, and western blot. RESULTS: Compared with the NC group, paw swelling, AI, IFN-gamma, and Th1/Th2 were increased, and pulmonary function parameters, IL-4, FoxP3 were decreased significantly in the MC group (P < 0.05 or P < 0.01). Pulmonary function parameters, Treg, IL-4, FoxP3 (and mRNA) were higher, and paw swelling, AI, and IFN-gamma (and mRNA) were lower in the XFC group than those in the MC group. The XFC group was also much better than the TPT group in improving pulmonary function, FoxP3 mRNA, IFN-gamma, IL-4, Th1/Th2, and IL-10 (P < 0.05 or P < 0.01). CONCLUSION: Xinfeng capsule can improve pulmonary function by regulating the levels of Tregs, inhibiting the activation of Th1 to Th2 cells, inducing drift, maintaining cell immune suppression, correcting the imbalance of Th1/Th2, and reducing inflammatory mediators.

Use of xinfeng capsule to treat abarticular pathologic changes in patients with rheumatoid arthritis.

OBJECTIVE: To observe the influence of Xinfengcapsule (XFC) on abarticular pathologic changes (APCs) and other indices of patients with rheumatoid arthritis (RA) and explore the mechanism of action of XFC in improving such changes. METHODS: Three-hundred RA patients were divided randomly into a treatment group (n = 150) and control group (n = 150). A normal control (NC) group (n = 90) was also created. Changes in cardiac function, pulmonary function, anemia indices and platelet parameters of RA patients were measured. Curative effects of the two groups were compared, and comparison carried out with the NC group. RESULTS: In 300 RA patients, late diastolic peak flow velocity (A peak) was much higher (P < 0.01) and early diastolic peak flow velocity (E peak), E/A, and left ventricular fraction shortening much lower(P < 0.01) than those in the NC group. Vital capacity (VC), forced vital capacity in one second, forced vitalcapacity (FVC), maximal voluntary ventilation (MVV), maximal expiratory flow in 50% of VC (FEF50) and FEF75 were lowered remarkably (P < 0.05 or P < 0.01). Platelet count (PLT), plateletcrit (PCT) and mean platelet volume (MPV) increased markedly (P < 0.05 or P < 0.01), and hemoglobin (Hb) level decreased significantly (P < 0.05). After XFC treatment, the A peak and PLT and PCT were much lower (P < 0.05), and E/A and the number of red blood cells as well as Hb level were much higher (P < 0.05), as were FVC, MVV and FEF50 (P < 0.05 or P < 0.01), in the treatment group than those in the NC group. Total score of pain and swelling in joints, uric-acid level and high-sensitivity C-reactive protein level were much lower, and superoxide dismutase level as well as the number of CD4 + CD25+ regulation T cells (Treg) and CD4+ CD25+ CD127- Treg were much higher (P < 0.05 or P < 0.01) in the treatment group than those in the NC group. CONCLUSION: RA patients with pathologic changes in joints also suffer from lower cardiac and pulmonary functions and from parameters of anemia and platelet factors. XFC can improve the symptoms of RA patients, ameliorate their cardiac and pulmonary functions and reduce the parameters of anemia and platelet factors. XFC lowers the immune inflammatory reaction to improve APCs in RA patients.

[Role of Notch-Jagged/Delta signaling pathway in arthritis rats of reduced lung function induced by adjuvant].

OBJECTIVE: To observe the changes of pulmonary function and Notch signaling pathway of lung tissues in adjuvant-induced arthritis rats, and to investigate the mechanism of reduced lung function. METHODS: A total of 30 rats were randomly divided into a normal group and a model group. Rats in the model group were induced to establish the adjuvant arthritis AA model by intradermally injecting 0.1 mL Freund's complete adjuvant into the right paw. After 30 days, we observed the paw edema volume, arthritis index, pulmonary function, histomorphology, and Notch receptor/ligand of the lung tissue. RESULTS: Compared with the normal group, the paw edema volume, arthritis index, average expiratory flow within 0.3 s (FEV0.3/FVC), and the level of Notch3, Notch4 and Jagged2 of the lung tissue in the model group was significantly increased, while maximum expiratory flow at 50% of vital capacity (FEF50), maximum expiratory flow at 75% of vital capacity (FEF75), forced expiratory flow (PEF) and the expression of Notch1 of Jagged1 and Delta1 in the lung were significantly decreased (P<0.05, P<0.01). There were significant positive correlations between FEV0.3/FVC and Notch4. FEV0.3/FVC, FEF25, FEF50 and Notch3, Delta1 were negatively correlated, respectively (P<0.05, P<0.01). CONCLUSION: While arthritis occurs in AA rats, pulmonary function declines and significantly correlates with the expression of Notch receptor/ligand. The deposition of immune complex in the lung after the injection of CFA activates the Notch signaling pathway, and results in further decline of pulmonary function by signaling cascades.

[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.