Exploring therapeutic targets for molecular therapy of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic and progressive interstitial lung disease with a poor prognosis. Idiopathic pulmonary fibrosis is characterized by repeated alveolar epithelial damage leading to abnormal repair. The intercellular microenvironment is disturbed, leading to continuous activation of fibroblasts and myofibroblasts, deposition of extracellular matrix, and ultimately fibrosis. Moreover, pulmonary fibrosis was also found as a COVID-19 complication. Currently, two drugs, pirfenidone and nintedanib, are approved for clinical therapy worldwide. However, they can merely slow the disease's progression rather than rescue it. These two drugs have other limitations, such as lack of efficacy, adverse effects, and poor pharmacokinetics. Consequently, a growing number of molecular therapies have been actively developed. Treatment options for IPF are becoming increasingly available. This article reviews the research platform, including cell and animal models involved in molecular therapy studies of idiopathic pulmonary fibrosis as well as the promising therapeutic targets and their development progress during clinical trials. The former includes patient case/control studies, cell models, and animal models. The latter includes transforming growth factor-beta, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid, interleukin-13, Rho-associated coiled-coil forming protein kinase family, and Janus kinases/signal transducers and activators of transcription pathway. We mainly focused on the therapeutic targets that have not only entered clinical trials but were publicly published with their clinical outcomes. Moreover, this work provides an outlook on some promising targets for further validation of their possibilities to cure the disease.

SLC38A6 expression in macrophages exacerbates pulmonary inflammation.

Pulmonary inflammation involves complex changes of the immune cells, in which macrophages play important roles and their function might be influenced by metabolism. Slc38a6 acts as a carrier of nutrient for macrophages (Mφ) to exert the function. In this study, pneumonia patient blood was found up-regulated SLC38A6 expression, which correlated with monocytes number and white blood cell number. The similar result was also shown in LPS induced sepsis mice. To reveal the key role of Slc38a6, we used systemic and conditional knock-out mice. Either systemic or LyzCRE specific knock-out could alleviate the severity of sepsis mice, reduce the proinflammatory cytokine TNF-α and IL-1ß expression in serum and decrease the monocytes number in bronchial alveolar lavage and peritoneal lavage via flow cytometry. In order to reveal the signal of up-regulated Slc38a6, the Tlr4 signal inhibitor TAK242 and TLR4 knock-out mice were used. By blocking Tlr4 signal in macrophages via TAK242, the expression of Slc38a6 was down-regulated synchronously, and the same results were also found in Tlr4 knock-out macrophages. However, in the overexpressed Slc38a6 macrophages, blocking Tlr4 signal via TAK242, 20% of the mRNA expression of IL-1ß still could be expressed, indicating that up-regulated Slc38a6 participates in IL-1ß expression process. Collectively, it is the first time showed that an amino acid transporter SLC38A6 up-regulated in monocytes/macrophages promotes activation in pulmonary inflammation. SLC38A6 might be a promising target molecule for pulmonary inflammation treatment.

An update on Ym1 and its immunoregulatory role in diseases.

Ym1 is a rodent-specific chitinase-like protein (CLP) lacking catalytic activity, whose cellular origins are mainly macrophages, neutrophils and other cells. Although the detailed function of Ym1 remains poorly understood, Ym1 has been generally recognized as a fundamental feature of alternative activation of macrophages in mice and hence one of the prevalent detecting targets in macrophage phenotype distinguishment. Studies have pointed out that Ym1 may have regulatory effects, which are multifaceted and even contradictory, far more than just a mere marker. Allergic lung inflammation, parasite infection, autoimmune diseases, and central nervous system diseases have been found associations with Ym1 to varying degrees. Thus, insights into Ym1's role in diseases would help us understand the pathogenesis of different diseases and clarify the genuine roles of CLPs in mammals. This review summarizes the information on Ym1 from the gene to its expression and regulation and focuses on the association between Ym1 and diseases.

A shedding soluble form of interleukin-17 receptor D exacerbates collagen-induced arthritis through facilitating TNF-α-dependent receptor clustering.

Rheumatoid arthritis (RA) is exacerbated by TNF-alpha signaling. However, it remains unclear whether TNF-α-activated TNFR1 and TNFR2 are regulated by extracellular factors. Here, we showed that soluble glycosylated interleukin-17 receptor D (sIL-17RD), which was produced by proteolytic cleavage, enhanced TNF-α-induced RA. We revealed that IL-17RD shedding was induced by the proteolytic enzyme TACE and enhanced by TNF-α expression in macrophages. Intriguingly, sIL-17RD was elevated in the sera of arthritic mice and rats. Recombinant sIL-17RD significantly enhanced the TNF-α-induced proinflammatory response by promoting TNF-α-TNFR-sIL-17RD complex formation and receptor clustering, leading to the accelerated development of collagen-induced arthritis. Our observations revealed that ectodomain shedding of IL-17RD occurred in RA to boost the TNF-α-induced inflammatory response. Targeting sIL-17RD may provide a new strategy for the therapy of RA.

Pristane promotes anaerobic glycolysis to facilitate proinflammatory activation of macrophages and development of arthritis.

Pristane-induced arthritis (PIA) could be adoptively transferred by splenic T cells in rats, and innate immunity should play critical roles in T cell activation. However, in pre-clinical stage, the activation mechanism of innate cells like macrophages remains unclear. Here we found that PIA was dependent on macrophages since cell depletion alleviated disease severity. Splenic macrophages of PIA rats showed M1 phenotypic shifting. The quantitative proteomics analysis suggested that macrophages initiated metabolic reprogramming with the conversion of aerobic oxidation to glycolysis in response to pristane in vivo. Notably, macrophages treated with pristane showed mitochondrial dysregulation and increased glycolysis flux and enzyme activity. Additionally, TNFα production, strongly associating with the glycolysis enzyme Ldha/Ldhb, could be reduced as glycolysis was inhibited or be enhanced as citrate cycle was blocked. This work provides detailed insights into the molecular mechanisms of pristane-mediated metabolic reprogramming in macrophages and suggests a new therapeutic strategy for arthritic disorders.

Up-regulated DERL3 in fibroblast-like synoviocytes exacerbates inflammation of rheumatoid arthritis.

Endoplasmic reticulum (ER) stress associated proteins contribute to the pathogenesis of rheumatoid arthritis (RA) through affecting synoviocyte proliferation and proinflammatory cytokine production. The role of DERL3, an ER-associated degradation component, in joint inflammation of RA was explored. Synovial tissues from RA and osteoarthritis (OA) patients were collected, and in RA synovial tissue, DERL3 showed up-regulation and significantly positive correlation with the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and matrix metalloproteinase (MMP)-1. Immunofluorescence result suggested DERL3 was located in fibroblast-like synoviocytes (FLS). Among different inflammatory stimuli, DERL3 could be up-regulated by TNF-α stimulation in FLS. Under TNF-α stimulation, knocking down DERL3, the expression of IL-6, IL-8, MMP-1, MMP-13 was reduced and the activation of nuclear factor kappa B (NF-κB) signaling pathway was inhibited. In pristane-induced arthritis (PIA) rat model, Derl3 was up-regulated in synovial tissue and disease was attenuated after intraarticular injection of siDerl3. Overall, we conclude that TNF-α inducing DERL3 expression promotes the inflammation of FLS through activation of NF-κB signaling pathway, suggesting DERL3 plays important roles in the pathogenesis of RA and is a promising therapeutic target.

Elevated IL-22 in psoriasis plays an anti-apoptotic role in keratinocytes through mediating Bcl-xL/Bax.

IL-22 is known to mediate inflammation in psoriasis, while IL-22 binding protein (IL-22BP) binds IL-22 to suppress IL-22 signaling. However, the function of IL-22 in regulating apoptosis in psoriasis remains poorly understood. In this study, we found that IL-22/IL-22R1 in lesional skin and IL-22 in serum from psoriatic patients were highly upregulated compared with healthy controls, while IL-22BP was not changed. Correlations between IL-22/IL-22R1 levels and the thickness of psoriatic lesions suggested that IL-22 might positively regulate abnormal hyperplasia in psoriasis. Apoptotic keratinocytes were increased only in stratum corneum, but not in spinous and basal layers of psoriasis. Moreover, IL-22 promoted cell viability in human epidermal keratinocytes (HEKs). The apoptosis induced by TNF-α and IFN-γ was inhibited in HEKs treated with IL-22, since that IL-22 upregulated Bcl-xL and downregulated Bax production in HEKs in the presence of TNF-α and IFN-γ. In addition, IL-22BP could counteract the anti-apoptotic effect of IL-22. Our finding demonstrates that IL-22 might play an anti-apoptosis role on keratinocytes to balance cell proliferation and apoptosis in psoriatic epidermis.

Upregulated PKM2 in Macrophages Exacerbates Experimental Arthritis via STAT1 Signaling.

Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1ß production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment.

Interpreting the MicroRNA-15/107 family: interaction identification by combining network based and experiment supported approach.

BACKGROUND: The highly conservative miR-15/107 family (also named as miR-15/107 gene group) including ten miRNA members is currently recognized strongly implicated in multiple human disorders. Some studies focus on the entire family rather than individual miRNA for a bigger picture, while there is also certain signature dysregulation for some of the individual miRNA implicated even in the same disorder. METHODS: Faced with the exponential growth of experimental evidence, our study tries to analyze their function and target interactions using various bioinformatics tools. RESULTS: Firstly, the evolutionary conservative "AGCAGC" sequence and possible clustered transcriptional pattern were described. Secondly, both the experimentally validated and bioinformatically predicted miRNA-target gene relationship of the entire family was analyzed to understand the mechanism of underlying collective effects for target regulation from the miR-15/107 family. Moreover, pathway analysis among miR-15/107 family was performed and displayed in detail, while its impact on cell proliferation is experimentally validated. Eventually, the dysregulation of miR-15/107 in diseases was discussed. CONCLUSIONS: In summary, our study proposes that the collective functions and implication of miR-15/107 family in various human diseases are achieved relying on the massive overlapping target genes. While the minor differences within target gene interaction among family members could also explain the signature behavior for some of the individual miRNA in aspects such as its disease-specific dysregulation and various participation in pathways.

Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.

Down-regulation of miR-10a-5p promotes proliferation and restricts apoptosis via targeting T-box transcription factor 5 in inflamed synoviocytes.

Synoviocytes from rheumatoid arthritis (RA) patients share certain features with tumor cells, such as over proliferation and invasion. Anomalous microRNA (miRNA) expression may participate in the pathogenesis of RA in different ways. The objective of the present study was to observe the role of miR-10a-5p targeting T-box transcription factor 5 (TBX5) gene on synoviocyte proliferation and apoptosis in RA. Human synovial sarcoma cell line, SW982 cells stimulating with interleukin-1ß (IL-1ß) were transfected with miR-10a-5p mimic and siRNA of TBX5. The real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to evaluate the expression level of miR-10a-5p and TBX5 in SW982 cells respectively. Further, the proliferation and apoptosis of SW982 cells after treatment were determined by cell counting kit (CCK-8) and flow cytometry analysis respectively. We found that the miR-10a-5p showed down-regulated while TBX5 showed up-regulated expression in synoviocytes after stimulation with IL-1ß. The miR-10a-5p mimic treatment showed a decline in cell proliferation while the increased rate of cell apoptosis as compared with control. Moreover, knockdown of TBX5 favored the apoptosis and reduced the cell proliferation as compared with control group. We conclude that down-regulation of miR-10a-5p promotes proliferation and restricts apoptosis via targeting TBX5 in inflamed synoviocytes.

Down-regulation of miR-10a-5p in synoviocytes contributes to TBX5-controlled joint inflammation.

MicroRNAs are considered to play critical roles in the pathogenesis of human inflammatory arthritis, including rheumatoid arthritis (RA). The purpose of this study was to determine the relationship between miR-10a-5p and TBX5 in synoviocytes and evaluate their contribution to joint inflammation. The expression of miR-10a-5p and TBX5 in the synovium of RA and human synovial sarcoma cell line SW982 stimulated by IL-1ß was determined by RT-qPCR and Western blotting. The direct interaction between miR-10a-5p and TBX5 3'UTR was determined by dual-luciferase reporter assay in HeLa cells. Mimics and inhibitors of miR-10a-5p were transfected into SW982 cells. TBX5 was overexpressed by plasmid transfection or knocked down by RNAi. Proinflammatory cytokines and TLR3 and MMP13 expressions were determined by RT-qPCR and Western blotting. Down-regulated expression of miR-10a-5p and up-regulation of TBX5 in human patients with RA were found compared to patients with OA. IL-1ß could reduce miR-10a-5p and increase TBX5 expression in SW982 cells in vitro. The direct target relationship between miR-10a-5p and 3'UTR of TBX5 was confirmed by luciferase reporter assay. Alterations of miR-10-5p after transfection with its mimic and inhibitor caused the related depression and re-expression of TBX5 and inflammatory factors in SW982 cells. Overexpression of TBX5 after pCMV3-TBX5 plasmid transfection significantly promoted the production of TLR3, MMP13 and various inflammatory cytokines, while this effect was rescued after knocking down of TBX5 with its specific siRNA. We conclude that miR-10a-5p in a relation with TBX5 regulates joint inflammation in arthritis, which would serve as a diagnostic and therapeutic target for RA treatment.

Bioinformatics analysis of genetic variants of endoplasmic reticulum aminopeptidase 1 in ankylosing spondylitis.

According to the results of the first genome-wide association study of ankylosing spondylitis (AS), endoplasmic reticulum aminopeptidase 1 (ERAP1) may serve an important role. However, a number of case-control studies have not been able to replicate this result using the same genetic markers. In the present study, the role of common genetic variants of ERAP1 in AS was investigated using two-stage bioinformatics analysis. In the first stage, a classical meta-analysis was performed to assess AS susceptibility markers in ERAP1 using data from available published case-control association studies. The summary odds ratios for 10 single nucleotide polymorphisms (SNPs) were observed to be statistically significant in different studies. In the second stage, the functional effects of these genetic ERAP1 variants were investigated using prediction tools and structural analyses. The K528R (rs30187) substitution SNP in ERAP1 was termed as likely damaging by PolyPhen-2 software, was observed to be located close to the entrance of the substrate pocket, and was predicted to contribute to reduced ERAP1 aminopeptidase activity. In addition, the R725Q (rs17482078) SNP, which was an additional potentially damaging substitution, was suggested to decrease the enzymatic activity of ERAP1, as this substitution may lead to the loss of two hydrogen bonds between R725 and D766 and affect the stability of the C-terminus of ERAP1. In conclusion, the results of the two-stage bioinformatics analysis supported the hypothesis that ERAP1 may present an important susceptibility gene for AS. In addition, the results revealed that two functional SNPs (rs30187 and rs17482078) demonstrated the potential to decrease the enzymatic activity of ERAP1 by affecting its protein structure. Further protein structure-guided studies of the specificity and activity of these ERAP1 variants are therefore warranted.

Pristane induces autophagy in macrophages, promoting a STAT1-IRF1-TLR3 pathway and arthritis.

Autophagy is involved in both innate and adaptive immune regulation. We propose that autophagy regulates activation of TLR3 in macrophages and is thereby essential for development of pristane-induced arthritis. We found that pristane treatment induced autophagy in macrophages in vitro and in vivo, in spleen cells from pristane injected rats. The induced autophagy was associated with STAT1 phosphorylation and expression of IRF1 and TLR3. Blocking the pristane activated autophagy by Wortmannin and Bafilomycin A1 or by RNAi of Becn1 led to a downregulation of the associated STAT1-IRF1-TLR3 pathway. Most importantly, the development of arthritis was alleviated by suppressing either autophagy or TLR3. We conclude that pristane enhanced autophagy, leading to a STAT1-IRF1 controlled upregulation of TLR3 expression in macrophages, is a pathogenic mechanism in the development of arthritis.

Discovery of 4-benzoylamino-N-(prop-2-yn-1-yl)benzamides as novel microRNA-21 inhibitors.

MicroRNA-21, as an oncogenic miRNA, has caught great attention for medicinal chemists to develop its novel inhibitors for cancer therapy. In the present study, we designed 4-benzoylamino-N-(prop-2-yn-1-yl)benzamides as miR-21 inhibitor candidates on the basis of scaffold hopping. Eighteen compounds were synthesized. The inhibitory activities of synthesized compounds against the expression of miR-21 were evaluated using stem loop RT-qPCR and compound 1j was discovered as the most potent compound, which displayed a time and concentration dependent inhibition manner. In addition, various functional assays such as the expression of miR-21 target gene detected by Western blotting and the cell growth and apoptosis detected by flow cytometric analysis were checked in Hela (human epithelioid cervix carcinoma) and U-87 MG (human glioblastoma) cells to confirm its activity. The results indicate that compound 1j can enhance apoptosis, retard proliferation, and up-regulate PDCD4, a target protein of miR-21. In addition, the compound 1j does not influence the expression of multiple miRNAs and the genes that participate in miRNA universal biosynthesis pathway. These results strongly support the assumption that title compounds can serve as a small molecule inhibitor of miR-21.

Pristane primed rat T cells enhance TLR3 expression of fibroblast-like synoviocytes via TNF-α initiated p38 MAPK and NF-κB pathways.

Based on pristane-induced arthritis (PIA), we found that T cells mediate TLR3 overexpression in fibroblast-like synoviocytes (FLS). The aim of this study is to determine key factors by which T cells induce TLR3 expression. Rat FLS were co-cultured with pristane primed T cell conditioned medium (PPT medium), and TLR3 expression of FLS was significantly induced. TNF-α, IFN-γ and IL-17 were dominantly expressed in PIA T cells. The overexpression of TLR3 and its related genes in FLS co-cultured with PPT medium could be reduced through blocking TNF-α pathway. CD4(+) T cells from spleen of PIA rats showed increase of TNF-α secretion. P38 MAPK and NF-κB were activated in FLS by PPT medium, and their inhibitors decreased TLR3 upregulation significantly. Finally, TNF-α induced TLR3 expression was confirmed in human synovial cells. Summarily, TNF-α derived from pristane primed T cells induced TLR3 expression of FLS through activating p38 MAPK and NF-κB pathways.

The elevated expression of Th17-related cytokines and receptors is associated with skin lesion severity in early systemic sclerosis.

OBJECTIVE: The objective was to survey the expression and localization of Th17-related cytokines and their correlation with skin lesion severity in early systemic sclerosis (SSc). METHODS: The mRNA expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR) from 21 SSc patients and 12 healthy controls (HC). The protein expression was examined by immunohistochemistry (IHC) and Western blotting. RESULTS: The RT-qPCR analysis showed a significantly higher expression of IL-17A, IL-21, IL-22, IL-26, IL-17RA, IL-21R, and IL-22R1 mRNA; consistently, the IHC analysis showed an over-expression of IL-17RA, IL-21R and IL-22R1 and the Western blotting analysis showed an over-expression of IL-17A, IL-21, IL-21R and IL-22R1 in early SSc skin lesions. The mRNA levels of IL-21 were higher in diffuse cutaneous than limited cutaneous SSc lesions. The mRNA expression of IL-26, IL-22, IL-22R1, mRNA and protein expression of IL-17A, IL-21, IL-21R were positively correlated with the modified Rodnan skin score of SSc. In addition, the mRNA levels of ICAM-1 were positively correlated with IL-17A/IL-17RA, and VEGFA and IL-4 were both positively correlated with IL-21/IL-21R, while TGF-ß were moderately negatively correlated with IL-22/IL-22R1. CONCLUSIONS: Th17 cytokines contribute to progression in early SSc skin lesions. IL-21/IL-21R could act as potential biomarkers presenting early SSc skin lesions severity.

MicroRNA-26a negatively regulates toll-like receptor 3 expression of rat macrophages and ameliorates pristane induced arthritis in rats.

INTRODUCTION: Abnormal toll-like receptor (TLR)3 signaling plays an indispensable role in pathogenesis of both experimental and human rheumatoid arthritis, and microRNAs (miRNAs) might orchestrate this signaling pathway. This study was performed to determine the relationship between miR-26a and TLR3 in rat macrophages and to observe effects of miR-26a mimic on pristane induced arthritis (PIA) in rats. METHODS: Dual luciferase reporter assay was used to validate the direct interaction between miR-26a (a candidate miRNA to target tlr3 mRNA) and tlr3 3'UTR. MiR-26a regulation on TLR3 gene expression was determined using RT-qPCR and Western blotting after miR-26a mimics and inhibitors were transfected into rat macrophage line NR8383 cells. Poly I:C (TLR3 ligand) was used to trigger TLR3 activation, and mRNA expression of its downstream cytokines interferon (ifn)-ß and tumor necrosis factor (tnf)-α was accordingly detected to determine the regulation of TLR3 signaling. Expressions of TLR3 and miR-26a were detected during rat bone marrow derived macrophage (BMDM) induction, in pristane stimulated NR8383 cells and spleens from methotrexate (MTX) treated PIA rats. A miR-26a mimic was administrated intraperitoneally to PIA rats, and arthritis severity was evaluated by macroscopic or microscopic observations. RESULTS: Direct target relationship between miR-26a and tlr3 mRNA in rats was confirmed. Modifications of miR-26a function by transfection of miR-26a mimics and inhibitors exhibited corresponding repression and augmentation of TLR3 and its signaling downstream cytokine expressions in NR8383 cells. The alteration of miR-26a expression was negatively related with TLR3 expression during BMDM induction, in pristane-primed NR8383 cells and PIA rat spleens. Moreover, both abnormal expressions were rescued in MTX treated arthritis rat spleens. The miR-26a mimic treatment displayed the depression of TLR3 expression and ameliorated the disease severity in the rats with pristane induced arthritis. CONCLUSIONS: MiR-26a negatively regulates TLR3 signaling via targeting of TLR3 itself in rat macrophages, and this finding provides a novel insight into abnormal TLR3 overexpression during experimental arthritis.

Methotrexate ameliorates pristane-induced arthritis by decreasing IFN-γ and IL-17A expressions.

OBJECTIVE: This study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats. METHODS: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane, and then treated with MTX or BSO. Arthritis severity was evaluated macroscopically and microscopically. Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The clinical arthritis severity was decreased after MTX treatment, while the BSO groups did not show significant changes compared with the disease group. The plasma NO level of the MTX group was significantly decreased compared with the disease group, but the BSO groups showed no difference from the disease group in plasma NO levels. The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group, but only showed a declining trend in the BSO groups compared with the disease group. Neither MTX nor BSO had an effect on the mRNA expressions of IL-4, transforming growth factor ß (TGF-ß), and tumor necrosis factor-α (TNF-α) in the spleen. CONCLUSIONS: MTX, but not BSO, can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.

Toll-like receptor 3 upregulation in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats.

INTRODUCTION: Toll-like receptors (TLRs) are involved in both innate and adaptive immune responses and are likely to play a complex role in the pathogenesis of human rheumatoid arthritis (RA) and experimental arthritis. The objective of this study was to identify the key TLR in pristane-induced arthritis (PIA), a rat model for RA, and to clarify its roles in the initiation and maintenance of arthritis. METHODS: Arthritis in DA rats was induced by pristane and the severity was evaluated by macroscopic and microscopic score systems. Spleen TLR and cytokine expression was detected at different time points by real-time polymerase chain reaction (PCR) and flow cytometry. Polyinosine-polycytidylic acid (polyI:C, a ligand of TLR3) or TLR3 specific short-hairpin RNA plasmid for RNA interference was administrated to PIA rats in vivo. Serum nitrogen oxide concentration was determined by Griess method, and tumor necrosis factor alpha (TNF-alpha) was determined by L929 biotest. In splenic macrophages, TLR3 expression was measured by flow cytometry. A rat macrophage cell line (NR8383) was stimulated by pristane, and anti-TLR3 antibody were used to block TLR3 pathway. TLR3 and cytokine expression in NR8383 were detected by real-time PCR. RESULTS: By screening the TLR expression profile in spleen of DA rats after pristane injection, we found that TLR3 was the most early and prominently upregulated TLR. Both TLR3 mRNA and protein expression of spleen were upregulated at 6 and 26 days after pristane injection. Furthermore, administration of polyI:C exacerbated, whereas RNA interference targeting TLR3 ameliorated, the arthritis. Particularly, TLR3 expression was induced in splenic macrophages of PIA rats, and also in the NR8383 cell line after pristane stimulation in a dose- and time- dependent manner. Upregulation of interferon beta (IFN-beta) and TNF-alpha by pristane stimulation was blocked by anti-TLR3 antibody in NR8383. CONCLUSIONS: TLR3 plays a pivotal role in the initiation and development of PIA which may dependent on macrophage. These findings are useful to understand the pathogenesis of RA and may provide an intriguing therapeutic opportunity for RA.