Suppression of PGE2/EP2 signaling alleviates Hirschsprung disease by upregulating p38 mitogen-activated protein kinase activity.

Hirschsprung disease (HSCR) is a congenital disorder caused by the failure of enteric neural crest cells (ENCCs) to colonize the distal bowel, resulting in absence of enteric nervous system. While a range of molecules and signaling pathways have been found to contribute to HSCR development, the risk factors and pathogenesis of this disease in many patients remain unknown. We previously demonstrated that increased activity of the prostaglandin E2 (PGE2)/PGE2 receptor subtype EP2 pathway can be a risk factor for HSCR. In this study, an Ednrb-deficient mouse model of HSCR was generated and used to investigate if PGE2/EP2 pathway could be a potential therapeutic target for HSCR. We found that downregulation of PGE2/EP2 signaling by siRNA-mediated ablation of a PGE2 synthase or pharmacologic blockage of EP2 enhanced ENCC colonization in the distal bowel of Ednrb-/- mice and alleviated their HSCR-like symptoms. Furthermore, blockage of EP2 was shown to promote ENCC migration through upregulating p38 mitogen-activated protein kinase activity, which was downregulated in the colon of Ednrb-/- mice and in the distal aganglionic bowel of HSCR patients. These data provide evidence that maternal exposure during embryonic development to an environment with dysregulated activation of the PGE2/EP2 pathway may predispose genetically susceptible offspring to HSCR, and avoidance or early disruption of maternal events (e.g. inflammation) that possibly enhance PGE2/EP2 signaling during pregnancy would reduce the occurrence and severity of this disease. KEY MESSAGES : Knockdown of PTGES alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling promotes ENCC migration via enhancing p38 activity.

mascRNA alleviates STING-TBK1 signaling-mediated immune response through promoting ubiquitination of STING.

mascRNA (MALAT1-associated small cytoplasmic RNA) is a tRNA-like cytoplasmic small noncoding RNA whose function remains elusive. We previously revealed that this small RNA negatively regulates TLR4/2-triggered proinflammatory response while positively regulates TLR3-induced antiviral response. Here, we investigated whether and how mascRNA influences the stimulator of interferon genes (STING) signaling-triggered immune response. We found that overexpression of mascRNA inhibited the expression of type I interferon (IFN) genes and proinflammatory cytokines in response to cytosolic DNA stimulation; meanwhile, the abundance of STING protein and the level of phosphorylated TBK1 and STAT1 was decreased. By contrast, depletion of mascRNA potentiated the expression of type I IFNs, increased STING protein abundance, and promoted STING-mediated phosphorylation of TBK1 and STAT1 in response to DNA stimulation. In a mouse model of DNA-induced lung injury, exogenous mascRNA mitigated the antiviral response and the severity of lung inflammation. Mechanically, mascRNA was found to promote STING for K48-linked ubiquitination and degradation in macrophages both with and without cytosolic DNA stimulation. Hence, mascRNA suppresses STING-TBK1 signaling-mediated innate immunity through promoting proteasomal degradation of STING, and this tRNA-like small RNA holds promise for the treatment of certain inflammatory diseases such as COVID-19 where aberrant STING signaling drives type I IFN immunopathology.

mascRNA promotes macrophage apoptosis, inhibits osteoclast differentiation and attenuates disease progression in a murine model of arthritis.

Macrophages play a crucial role in the pathogenesis of rheumatoid arthritis (RA) and have been considered as a therapeutic target of this disease. Here we show that mascRNA, a tRNA-like cytoplasmic small noncoding RNA, promoted RIPK1-dependent apoptosis (RDA) in RAW267.4 macrophages in response to the TAK1 inhibitor 5Z-7-oxozeaenol (5Z-7) alone as well as in combination with TNF. Moreover, mascRNA suppressed RANKL-induced expression of osteoclast marker genes and attenuated RANKL signaling. Using a murine model of collagen-induced arthritis (CIA), we demonstrated that mascRNA, administered either alone or in combination with 5Z-7, alleviated joint inflammation in CIA mice. Thus, mascRNA might be a promising agent for the treatment of RA.

The small RNA mascRNA differentially regulates TLR-induced proinflammatory and antiviral responses.

MALAT1-associated small cytoplasmic RNA (mascRNA) is a highly conserved transfer RNA-like (tRNA-like) noncoding RNA whose function remains largely unknown. We show here that this small RNA molecule played a role in the stringent control of TLR-mediated innate immune responses. mascRNA inhibited activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling and the production of inflammatory cytokines in macrophages stimulated with LPS, a TLR4 ligand. Furthermore, exogenous mascRNA alleviated LPS-induced lung inflammation. However, mascRNA potentiated the phosphorylation of IRF3 and STAT1 and the transcription of IFN-related genes in response to the TLR3 ligand poly(I:C) both in vitro and in vivo. Mechanistically, mascRNA was found to enhance K48-linked ubiquitination and proteasomal degradation of TRAF6, thereby negatively regulating TLR-mediated MyD88-dependent proinflammatory signaling while positively regulating TRIF-dependent IFN signaling. Additionally, heterogeneous nuclear ribonucleoprotein H (hnRNP H) and hnRNP F were found to interact with mascRNA, promote its degradation, and contribute to the fine-tuning of TLR-triggered immune responses. Taken together, our data identify a dual role of mascRNA in both negative and positive regulation of innate immune responses.

ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer.

ABIN-1, also called TNIP1, is an ubiquitin-binding protein that serves an important role in suppressing RIPK1-independent apoptosis, necroptosis, and NF-κB activation. However, the involvement of ABIN-1 in the regulation of RIPK1-dependent apoptosis (RDA) is unknown. In this study, we found that poly(I:C) + TAK1 inhibitor 5Z-7-oxozeaenol (P5) concurrently induces RDA and necroptosis in Abin-1-/-, but not in Abin-1+/+ mouse embryonic fibroblasts (MEFs). Upon P5 stimulation, cells initially die by necroptosis and subsequently by RDA. Furthermore, we explored the therapeutic effect of ABIN-1 deficiency in necroptosis-based cancer therapy in colorectal cancer (CRC). We found that poly(I:C) + 5Z-7-oxozeaenol + IDN-6556 (P5I) yields a robust pro-necroptosis response, and ABIN-1 deficiency additionally enhances this P5I-induced necroptosis. Moreover, phase I/II cIAP inhibitor birinapant with clinical caspase inhibitor IDN-6556 (BI) alone and 5-fluorouracil with IDN-6556 (FI) alone are sufficient to induce necroptotic cell death in CRC cells by promoting auto-secretion of tumor necrosis factor (TNF); ABIN-1 deficiency amplifies the BI- or FI-induced necroptosis. Two independent xenograft experiments using HT-29 or COLO205 cells show that both BI and P5I remarkably inhibit tumor growth via necroptosis activation. For poly(I:C)-induced cell death, the sensitizing effect of ABIN-1 deficiency on cell death may be attributed to increased expression of TLR3. In TNF-induced necroptosis, ABIN-1 deficiency increases TNF-induced RIPK1 polyubiquitination by reducing the recruitment of ubiquitin-editing enzyme A20 to the TNFR1 signaling complex and induces more TNF secretion in CRC cells upon pro-necroptosis stimulation. With this combined data, ABIN-1 deficiency promotes greater sensitization of CRC cells to necroptosis.

Multiple 'omics'-analysis reveals the role of prostaglandin E2 in Hirschsprung's disease.

The etiology and pathogenesis of Hirschsprung's disease (HSCR) remain largely unknown. We examined colon tissues from three independent populations with a combined analysis of metabolomics, transcriptomics and proteomics to understand HSCR pathogenesis, according to which mouse model was used to examine prostaglandin E2 (PGE2) induced clinical presentation of HSCR. SH-SY5Y and SK-N-BE(2) cell lines were studied for PGE2 inhibited cell migration through EP2. Our integrated multiple 'omics'-analysis suggests that the levels of PGE2, the expression of the gene encoding PGE2 receptor (EP2), and PGE2 synthesis enzyme genes (PTGS1 and PTGES) increased in HSCR colon tissues, together with a decreased synthesis of PGE2-related byproducts. In vivo, the pregnant mice treated with PGE2 gave birth to offspring with the decrease of ganglion cells in their colon and gut function. In in vitro study, when EP2 was blocked, the PGE2-inhibited cell migration was recovered. Our study identified a novel pathway highlighting the link between expression of PTGS1 and PTGES, levels of PGE2, expression of PTGER2, and neural crest cell migration in HSCR, providing a novel strategy for future diagnosis and prevention of HSCR.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Retrospective study of the prognostic significance of neutrophil-to-lymphocyte ratio for postsurgical outcomes of patients with endometrial carcinoma.

OBJECTIVE: To evaluate the significance of postoperative inflammatory system response markers in predicting the prognosis of patients with endometrial cancer undergoing surgery. METHODS: The present retrospective study included patients who underwent surgical treatment for pathology-confirmed endometrial cancer between January 1, 2007, and June 30, 2013, at the Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. Potential prognostic factors were investigated by Cox proportional hazards analysis and survival rates were compared using Kaplan-Meier analyses. RESULTS: There were 185 patients with surgically treated endometrial cancer included. Multivariate analyses demonstrated that a preoperative neutrophil-to-lymphocyte ratio (NLR) above 1.81 (P=0.010) and a postoperative NLR above 7.54 (P=0.008) were both independently associated with lower disease free survival. Elevated preoperative and postoperative NLRs were associated with higher tumor stage (P=0.021 and P=0.009, respectively), and only elevated preoperative NLR was associated with lymph node involvement (P=0.023). CONCLUSION: Preoperative and postoperative NLRs were independently associated with inflammatory system response markers and could be combined to evaluate the prognosis of patients with endometrial cancer following surgery.

Long non-coding RNA: a versatile regulator of the nuclear factor-κB signalling circuit.

The nuclear factor-κB (NF-κB) family of transcription factors play an essential role for the regulation of inflammatory responses, immune function and malignant transformation. Aberrant activity of this signalling pathway may lead to inflammation, autoimmune diseases and oncogenesis. Over the last two decades great progress has been made in the understanding of NF-κB activation and how the response is counteracted for maintaining tissue homeostasis. Therapeutic targeting of this pathway has largely remained ineffective due to the widespread role of this vital pathway and the lack of specificity of the therapies currently available. Besides regulatory proteins and microRNAs, long non-coding RNA (lncRNA) is emerging as another critical layer of the intricate modulatory architecture for the control of the NF-κB signalling circuit. In this paper we focus on recent progress concerning lncRNA-mediated modulation of the NF-κB pathway, and evaluate the potential therapeutic uses and challenges of using lncRNAs that regulate NF-κB activity.

The long noncoding RNA MALAT1 regulates the lipopolysaccharide-induced inflammatory response through its interaction with NF-κB.

MALAT1 is a conserved long noncoding RNA whose expression correlates with many human cancers. However, its significance in immunity remains largely unknown. Here, we observe that MALAT1 is upregulated in lipopolysaccharide (LPS)-activated macrophages. Knockdown of MALAT1 increases LPS-induced expression of TNFα and IL-6. Mechanistically, MALAT1 was found to interact with NF-κB in the nucleus, thus inhibiting its DNA binding activity and consequently decreasing the production of inflammatory cytokines. Additionally, abnormal expression of MALAT1 was found to be NF-κB-dependent. These findings suggest that MALAT1 may function as an autonegative feedback regulator of NF-κB to help fine-tune innate immune responses.

A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus.

A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone-usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5'-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated. Expression of MXAN3487 was not affected by sulphate availability, suggesting that MXAN3487 may not function in a reductive sulphate assimilation pathway. Deletion of MXAN3487 significantly delayed fruiting body formation and the production of McuA, a spore coat protein secreted by the M. xanthus Mcu CU system. Based on these observations and data from our previous studies, we propose that MXAN3487 may phosphorylate molecules structurally related to APS, generating metabolites necessary for M. xanthus development, and that MXAN3487 exerts a positive effect on the mcuABC operon whose expression is morphogenesis dependent.

Identification of a putative flavin adenine dinucleotide-binding monooxygenase as a regulator for Myxococcus xanthus development.

UNLABELLED: Gene clusters coding for the chaperone/usher (CU) pathway are widely distributed in many important environmental and pathogenic microbes; however, information about the regulatory machineries controlling CU gene expression during multicellular morphogenesis is missing. The Myxococcus xanthus Mcu system, encoded by the mcuABCD gene cluster, represents a prototype of the archaic CU family that functions in spore coat formation. Using genome-wide transposon mutagenesis, we identified MXAN2872 to be a potential regulator of the mcuABC operon and demonstrated the necessity of MXAN2872 for mcuABC expression and fruiting body morphogenesis in early development. In silico, biochemical, and genetic analyses suggest that MXAN2872 encodes a Baeyer-Villiger monooxygenase (BVMO) of flavoproteins, and the potential cofactor-binding site as well as the BVMO fingerprint sequence is important for the regulatory role of the MXAN2872 protein. The expression profile of mcuA in strains with an MXAN2872 deletion and point mutation agrees well with the timing of cell aggregation of these mutants. Furthermore, McuA could not be detected either in a fruA-null mutant, where starvation-induced aggregation was completely blocked, or in the glycerol-induced spores, where sporulation was uncoupled from cell aggregation. In sum, the present work uncovers a positive role for MXAN2872, a metabolic enzyme-encoding gene, in controlling M. xanthus development. MXAN2872 functions by affecting the onset of cell aggregation, thereby leading to a secondary effect on the timing of mcuABC expression of this model organism. IMPORTANCE: Identification of the players that drive Myxococcus xanthus fruiting body formation is necessary for studying the mechanism of multicellular morphogenesis in this model organism. This study identifies MXAN2872, a gene encoding a putative flavin adenine dinucleotide-binding monooxygenase, to be a new interesting regulator regulating the timing of developmental aggregation. In addition, MXAN2872 seems to affect the expression of the chaperone/usher gene cluster mcu in a manner that is aggregation dependent. Thus, in organisms characterized by a developmental cycle, expression of the chaperone/usher pathway can be controlled by morphological checkpoints, demonstrating another layer of complexity in the regulation of this conserved protein secretion pathway.

[The establishment, development and application of classification approach of freshwater phytoplankton based on the functional group: a review].

Appropriate schemes for classification of freshwater phytoplankton are prerequisites and important tools for revealing phytoplanktonic succession and studying freshwater ecosystems. An alternative approach, functional group of freshwater phytoplankton, has been proposed and developed due to the deficiencies of Linnaean and molecular identification in ecological applications. The functional group of phytoplankton is a classification scheme based on autoecology. In this study, the theoretical basis and classification criterion of functional group (FG), morpho-functional group (MFG) and morphology-based functional group (MBFG) were summarized, as well as their merits and demerits. FG was considered as the optimal classification approach for the aquatic ecology research and aquatic environment evaluation. The application status of FG was introduced, with the evaluation standards and problems of two approaches to assess water quality on the basis of FG, index methods of Q and QR, being briefly discussed.

The archaic chaperone-usher pathways may depend on donor strand exchange for intersubunit interactions.

Subunit-subunit interactions of the classical and alternate chaperone-usher (CU) systems have been shown to proceed through a donor strand exchange (DSE) mechanism. However, it is not known whether DSE is required for intersubunit interactions in the archaic CU system. We have previously shown that the Myxococcus xanthus Mcu system, a member of the archaic CU family that functions in spore coat formation, is likely to use the principle of donor strand complementation to medicate chaperone-subunit interactions analogous to the classical CU pathway. Here we describe the results of studies on Mcu subunit-subunit interactions. We constructed a series of N-terminal-deleted, single amino acid-mutated and donor strand-complemented Mcu subunits, and characterized their abilities to participate in subunit-subunit interactions. It appears that certain residues in both the N and C termini of McuA, a subunit of the Mcu system, play a critical role in intersubunit interactions and these interactions may involve the general principle of DSE of the classical and alternate CU systems. In addition, the specificity of the M. xanthus CU system for Mcu subunits over other spore coat proteins is demonstrated.

Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis.

AIMS: This study aimed to examine the efficacy of a nanocarrier (polyethyleneimine [PEI]-superparamagnetic iron oxide nanoparticle [SPIO]), composed of a core of iron oxide and a shell of PEI, in the systemic delivery of therapeutic siRNA to experimental arthritic joints. MATERIALS & METHODS: PEI-SPIO/siRNA nanoparticles were synthesized and characterized in vitro. Nanoparticles were administered intravenously to arthritic rats to analyze cellular uptake, tissue distribution and the therapeutic effect of a siRNA against the IL-2/-15 receptor ß chain (IL-2/IL-15Rß). RESULTS: PEI-SPIOs loaded with siRNA displayed negligible cytotoxicity, improved siRNA stability, efficient uptake by macrophages and the ability to induce specific gene silencing in vitro. PEI-SPIO-delivered siRNA accumulated easily in inflamed joints and was efficiently taken up by joint macrophages and T cells. Although IL-2/IL-15Rß siRNA-loaded PEI-SPIOs alone were efficacious in the treatment of experimental arthritis, combination therapy with both PEI-SPIO/IL-2/IL-15Rß siRNA and a magnetic field displayed an additive anti-inflammatory effect. CONCLUSION: PEI-functionalized SPIOs can be employed for systemic siRNA delivery in rheumatoid arthritis and enhanced therapeutic benefit can be achieved by the use of an external magnetic field.

Systemic delivery of small interfering RNA targeting the interleukin-2/15 receptor ß chain prevents disease progression in experimental arthritis.

The role of interleukin (IL)-15 in the pathogenesis of rheumatoid arthritis (RA) is well established; however, systemic knockdown of IL-15 receptor (IL-15R) for reduction in inflammation at local sites has not been demonstrated. In this study, the therapeutic effect of intravenously administered siRNA targeting the ß chain of IL-15R which is shared by the receptor for IL-2 was examined in rats with adjuvant-induced arthritis (AA). Polyethylenimine (PEI)-complexed siRNA nanoparticles could easily accumulate in arthritic paws of AA rats. In the paws, the nanoparticles were avidly taken up by macrophages and to a lesser extent by T cells. Weekly administered IL-2/15Rß siRNA polyplexes were capable of decreasing disease progression in AA rats, with striking inhibition of clinical, radiologic, and histologic features of RA. The observed therapeutic effect was associated with reduced expression of proinflammatory mediators in the inflamed joints. Thus, this study provides evidence that IL-2/15Rß could be targeted for the treatment of RA.

Characterization of McuB, a periplasmic chaperone-like protein involved in the assembly of Myxococcus spore coat.

The MXAN3885 to -3882 gene locus cluster (designated here mcuABCD) of Myxococcus xanthus encodes a member of the archaic chaperone-usher (CU) systems that functions in spore coat formation. We show here that McuD, a putative spore coat protein, affects cellular accumulation and cell surface localization of the spore coat protein McuA. We previously reported that genetic disruption of the putative usher McuC nearly eliminates surface display of McuA and show here that lack of the periplasmic chaperone-like protein McuB, which forms a complex with McuA, has a similar effect. Deletion mutation confirms that the G1 ß strand of McuB is absolutely essential for the stability and secretion of McuA. Site-directed mutagenesis identified two additional alternating hydrophobic residues Ile113 and Val115, together with the highly conserved proline within the G1 strand, as critical residues for chaperone function. These findings suggest that the assembly proteins McuB and McuC mediate the transport of McuA onto the cell surface and that McuA may interact with another spore coat protein, McuD, for its secretion. Importantly, although our data argue that the M. xanthus CU system is likely to use the basic principle of donor strand complementation (DSC), as in the cases of classical CU pathways, to promote folding and stabilization of the structural subunit(s), the periplasmic chaperone McuB appears to exhibit structural variation in mediating chaperone-subunit interaction.

Evidence that a chaperone-usher-like pathway of Myxococcus xanthus functions in spore coat formation.

Many bacteria use the chaperone-usher (CU) secretion pathway to assemble on their surfaces typical or atypical fimbrial organelles. Four consecutive genes of Myxococcus xanthus DK1622, MXAN3885-3882, were predicted to constitute an operon encoding a CU-like system involved in the assembly of the spore coat; however, experimental evidence supporting this hypothesis was lacking. In this study, co-transcription of MXAN3885-3883 was verified, and we found that this operon was expressed 12-15 h after initiation of M. xanthus development under conditions of stringent starvation. The MXAN3885 protein, which is highly homologous to, but expressed earlier than, the spore coat protein U of another M. xanthus strain, DZF1, was present mainly on the outer surface of myxospores. Inactivation of MXAN3883, encoding a putative outer membrane usher, inhibited assembly of MXAN3885 protein on spore surfaces and caused certain morphological alterations in the spore coat. Hence, the CU-like pathway in M. xanthus indeed functions in spore coat biogenesis. Based on chaperone amino acid sequence comparisons, our analysis suggests that the structural basis of the M. xanthus CU-like pathway for spore coat assembly may be different from that of most surface structures assembled by classical CU systems.

Interleukin-15 receptor-directed immunotoxins atteunuate disease severity in rat adjuvant arthritis.

We previously constructed two Pseudomonas exotoxin (PE)-based immunotoxins, IL15-PEDelta293 (IT1) and IL15M-PEDelta293 (IT2), for eliminating interleukin-15 receptor (IL-15R)-overexpressing cells. These two immunotoxins were generated by fusing either wild-type human IL-15 or an antagonist mutant IL-15 (IL-15M) to a modified form of PE. In this study the anti-arthritic effect of IT1 and IT2 was investigated using the rat model of adjuvant arthritis (AA). We found that both IT1 and IT2 could specifically target IL-15R-positive cells and induce apoptosis. After AA induction, treatment with either IT1 or IT2 resulted in profound improvement of the disease, with reductions in levels of synovial mononuclear leukocytes and certain inflammatory factors. Clinical and histological comparisons, together with the analyses of mRNA expression and the signal transducer and activator of transcription-3 (STAT-3) phosphorylation, revealed that the two immunotoxins decreased joint inflammation in AA rats to a similar extent. These data suggest that eliminating IL-15R-bearing cells via immunotoxins may be a promising approach for RA treatment. In addition, wild-type IL-15-based immunotoxins can be as effective as antagonist mutant IL-15-based immunotoxins in preventing joint inflammation associated with rheumatoid arthritis.